Integrated nucleic acid diagnostic device

ABSTRACT

The present invention provides a miniaturized integrated nucleic acid diagnostic device and system. The device of the invention is generally capable of performing one or more sample acquisition and preparation operations, in combination with one or more sample analysis operations. For example, the device can integrate several or all of the operations involved in sample acquisition and storage, sample preparation and sample analysis, within a single integrated unit. The device is useful in a variety of applications, and most notably, nucleic acid based diagnostic applications and de novo sequencing applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/671,928, filed Jun. 27, 1996, now U.S. Pat. No. 5,922,591, which is acontinuation-in-part of U.S. patent application Ser. No. 08/589,027,filed Jan. 19, 1996, now U.S. Pat. No. 5,856,174, which claims thebenefit of U.S. Provisional application Ser. No. 60/000,703, filed Jun.29, 1995 and U.S. Provisional application Ser. No. 60/000,859, filedJul. 3, 1995. This application is also a continuation-in-part of U.S.patent application Ser. No. 09/210,025, filed Dec. 11, 1998, now U.S.Pat. No. 6,043,080.

GOVERNMENT RIGHTS

The present invention was made with U.S Government support under ATPGrant No. 70NANB5H1031. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

The relationship between structure and function of macromolecules if offundamental importance in the understanding of biological systems. Theserelationships are important to understanding, for example, the functionsof enzymes, structure of signalling proteins, ways in which cellscommunicate with each other, as well as mechanisms of cellular controland metabolic feedback.

Genetic information is critical in continuation of life processes. Lifeis substantially informationally based and its genetic content controlthe growth and reproduction of the organism. The amino acid sequences ofpolypeptides, which are critical features of all living systems, areencoded by the genetic material of the cell. Further, the properties ofthese polypeptides, e.g., as enzymes, functional proteins, andstructural proteins, are determined by the sequence of amino acids whichmake them up. As structure and function are integrally related, manybiological functions may be explained by elucidating the underlyingstructural features which provide those functions, and these structuresare determined by the underlying genetic information in the form ofpolynucleotide sequences. In addition to encoding polypeptides,polynucleotide sequences can also be specifically involved in, forexample, the control and regulation of gene expression.

The study of this genetic information has proved to be of great value inproviding a better understanding of life processes, as well asdiagnosing and treating a large number of disorders. In particular,disorders which are caused by mutations, deletions or repeats inspecific portions of the genome, may be readily diagnosed and/or treatedusing genetic techniques. Similarly, disorders caused by external agentsmay be diagnosed by detecting the presence of genetic material which isunique to the external agent, e.g., bacterial or viral DNA.

While current genetic methods are generally capable of identifying thesegenetic sequences, such methods generally rely on a multiplicity ofdistinct processes to elucidate the nucleic acid sequences, with eachprocess to introducing a potential for error into the overall process.These processes also draw from a large number of distinct disciplines,including chemistry, molecular biology, medicine and others. It wouldtherefore be desirable to integrate the various process used in geneticdiagnosis, in a single process, at a minimum cost, and with a maximumease of operation.

Interest has been growing in the fabrication of microfluidic devices.Typically, advances in the semiconductor manufacturing arts have beentranslated to the fabrication of micromechanical structures, e.g.,micropumps, microvalves and the like, and microfluidic devices includingminiature chambers and flow passages.

A number of researchers have attempted employ these microfabricationtechniques in the miniaturization of some of the processes involved ingenetic analysis in particular. For example, published PCT applicationSer. No. 94/05414, to Northrup and White, incorporated herein byreference in its entirety for all purposes, reports an integratedmicro-PCR apparatus for collection and amplification of nucleic acidsfrom a specimen. However, there remains a need for an apparatus whichcombines the various processing and analytical operations involved innucleic acid analysis. The present invention meets these and otherneeds.

SUMMARY OF THE INVENTION

The present invention generally provides miniature integrated fluidicsystems for carrying out a variety of preparative and analyticaloperations, as well as methods of operating these systems and methods ofusing these systems. In a first aspect, the present invention provides aminiature fluidic system which comprises a body having at least firstand second chambers disposed therein. Each of these first and secondchambers has a fluid inlet and is in fluid connection. At least one ofthese first and second chambers is a hybridization chamber for analyzinga component of a fluid sample. The hybridization chamber includes apolymer array which has a plurality of different polymer sequencescoupled to a surface of a single substrate, each of the plurality ofdifferent polymer sequences being coupled to the surface in a different,known location. The system further includes a sample inlet, fluidlyconnected to at least one of the first and second chambers, forintroducing a fluid sample into the system, and a fluid transport systemfor moving a fluid sample from the first chamber to the second chamber.

In a preferred aspect, the fluid direction system comprises a pneumaticmanifold for applying a differential pressure between the first chamberand the second chamber, to move said fluid sample for the first chamberto the second chamber.

In a related aspect, the present invention provides a miniature fluidicsystem, which is substantially the same as that described above, exceptthat in place or in addition to a hybridization chamber, the systemcomprises a separation channel for separating a component of said fluidsample. The separation channel is fluidly connected to at least one ofthe chambers and includes at least first and second electrodes inelectrical contact with opposite ends of the separation channel forapplying a voltage across said separation channel.

Similarly, in an additional aspect, the present invention provides asubstantially similar fluidic system as described, except where at leastone of the chambers comprises an in vitro transcription reactionchamber, the in vitro transcription reaction chamber having an effectiveamount of an RNA polymerase and four different nucleoside triphosphates,disposed therein.

Further, the system may comprise a body wherein at least one of thechambers is a cell lysis chamber which includes a cell lysis system, forlysing cells in said fluid sample.

In a still further related aspect, at least one of the chambers may be anucleic acid purification chamber, for separating nucleic acids in saidfluid sample from other contaminants in said fluid sample.

The present invention also provides a miniature fluidic system whichcomporises a differential pressure delivery system for transportingfluids through the system. In particular, in one aspect, the presentinvention provides a miniature fluidic system, which includes a bodyhaving at least a first reaction chamber fluidly connected to a secondreaction chamber by a fluid passage. The system also includes a sampleinlet, fluidly connected to the first chamber, for introducing a fluidsample into the system. The system further includes a differentialpressure delivery system for maintaining the first chamber at a firstpressure and the second chamber at a second pressure, wherein the firstpressure is greater than ambient pressure and the second pressure isgreater than said first pressure. When the second chamber is brought toambient pressure, the first pressure forces a liquid sample in the firstchamber into the second chamber.

In an alternate aspect, the fluidic system employs a differentialpressure delivery source for maintaining the first chamber at a firstpressure and the second chamber at a second pressure, where the secondpressure is less than ambient pressure and the first pressure is lessthan the second pressure. When the first chamber is brought to ambientpressure, the second pressure draws a liquid sample in the first chamberinto the second chamber.

The present invention also provides methods of directing, controllingand manipulating fluids in miniature of micro-fluidic systems.

For example, in one aspect, the present invention provides a method fordirecting a fluid sample in a miniature fluidic system which comprisesproviding a microfabricated device having at least first and secondchamber disposed therein, wherein each of said at least first and secondchambers is in fluid connection with a common chamber or channel, has atleast first and second controllable valves disposed across said fluidconnection, respectively, and includes at least one vent. The methodcomprises applying a positive pressure to the common chamber or channel.The at least first controllable valve is selectively opened, whereby thepositive pressure forces the fluid sample from the common chamber orchannel into the first chamber.

The method may further comprise applying a positive pressure to thefirst chamber and selectively opening the least first controllablevalve, whereby the positive pressure forces said fluid sample from theleast first chamber into the common chamber or channel.

The present invention also provides methods of mixing at least twodiscrete fluid components in a microfabricated fluidic system.Specifically, the method comprises providing a microfabricated channelhaving a vent disposed at an intermediate location in said channel.Typically, the vent includes a gas permeable, fluid barrier disposedacross the vent. At least two discrete fluid components are thenintroduced into the channel separated by a gas bubble. Upon flowing theat least two fluid components past the vent, the bubble will exit thevent, allowing the at least two fluid components to mix.

The present invention also provides methods of repeatedly measuring aknown volume of a fluid in a miniature fluidic system. In particular,the method comprises providing a microfabricated device having at leastfirst and second chambers disposed therein, wherein the at least firstand second chambers are in fluid connection, and wherein at least one ofthe chambers is a volumetric chamber having a known volume. Thevolumetric chamber is filled with the fluid to create a first aliquot ofthe fluid. This aliquot is then transported to the at least secondchamber and the filling and transporting steps are repeated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of a nucleic acid diagnosticsystem for analysis of nucleic acids from samples.

FIGS. 2A and 2b show schematic representations of two alternate reactionchamber designs from a cut-away view.

FIG. 3 shows a schematic representation of a miniature integrateddiagnostic device having a number of reaction chambers arranged in aserial geometry.

FIGS. 4A-C show a representation of a microcapillary electrophoresisdevice. FIGS. 4A and 4B show the microcapillary configured for carryingout alternate loading strategies for the microcapillary whereas FIG. 4Cillustrates the microcapillary in running mode.

FIG. 5A illustrates a top view of a miniature integrated device whichemploys a centralized geometry. FIG. 5B shows a side view of the samedevice wherein the central chamber is a pumping chamber, and employingdiaphragm valve structures for sealing reaction chambers.

FIGS. 6A and 6B schematic illustrations of pneumatic control manifoldsfor transporting fluid within a miniature integrated device. FIG. 6Ashows a manifold configuration suitable for application of negativepressure, or vacuum, whereas FIG. 6B shows a manifold configuration forapplication of positive pressures. FIG. 6C illustrates a pressureprofile for moving fluids among several reaction chambers.

FIG. 7A shows a schematic illustration of a reaction chamberincorporating a PZT element for use in mixing the contents of thereaction chamber. FIG. 7B shows mixing within a reaction chamberapplying the PZT mixing element as shown in FIG. 7A. FIG. 7C is a bargraph showing a comparison of hybridization intensities using mechanicalmixing, acoustic mixing, stagnant hybridization and optimized acousticmixing.

FIG. 8 is a schematic illustration of a side and top view of a base-unitfor use with a miniature integrated device.

FIG. 9 is a time temperature profile of thermal cycling in a miniaturereaction chamber and a display of the programmed cycling parameters.

FIG. 10A is a gel showing a time course of an RNA fragmentationreaction. FIG. 10B is a gel showing a comparison of the product of an invitro transcription reaction in a microchamber vs. a control (testtube). FIG. 10C is a comparison of the PCR product produced in a PCRthermal cycler and that produced by a microreactor.

FIG. 11 shows an embodiment of a reaction chamber employing anelectronic pH control system.

FIGS. 12A-C shows a schematic representation of a miniature integrateddevice employing a pneumatic fluid direction system utilizing a gaspermeable fluid barrier bound vents, e.g., a poorly wetting orhydrophobic membrane, and pneumatically controlled valves. FIG. 12Ashows an embodiment of a single chamber employing this system. FIG. 12Bis a schematic illustration of a debubbling chamber for linking discretefluid plugs that are separeted by a gas bubble. FIG. 12C schematicallyillustrates this system in an integrated device having numerouschambers, including degassing chamber, dosing or volumetric chamber,storage and reaction chambers. FIG. 12D is an illustration of aninjection molded substrate which embodies the system schematicallyillustrated in FIG. 12C.

FIG. 13 is a schematic representation of a device configuration forcarrying generic sample preparation reactions.

FIG. 14 is a schematic representation of a device configuration forcarrying multiple parallel reactions.

FIGS. 15A and 15B show a demonstration of integrated reactions in amicrofabricated polycarbonate device. FIG. 15A shows the layout of thedevice including the thermal configuration of the device. FIG. 15B showsthe results of PCR amplification and subsequent in vitro transcriptionwithin the chamber of the device.

DETAILED DESCRIPTION OF THE

INVENTION

I. General

It is a general object of the present invention to provide aminiaturized integrated nucleic acid diagnostic devices and systemsincorporating these devices. The device of the invention is generallycapable of performing one or more sample acquisition and preparationoperations, in combination with one or more sample analyis operations.For example, the device can integrate several or all of the operationsinvolved in sample acquisition and storage, sample preparation andsample analysis, within a single, miniaturized, integrated unit. Thedevice is useful in a variety of applications and most notably, nucleicacid based diagnostic applications and de novo sequencing applications.

The device of the invention will typically be one component of a largerdiagnostic system which further includes a reader device for scanningand obtaining the data from the device, and a computer based interfacefor controlling the device and/or interpretation of the data derivedfrom the device.

To carry out its primary function, one embodiment of the device of theinvention will typically incorporate a plurality of distinct reactionchambers for carrying out the sample acquisition, preparation andanalysis operations. In particular, a sample to be analyzed isintroduced into the device whereupon it will be delivered to one ofthese distinct reaction chambers which are designed for carrying out avariety of reactions as a prelude to analysis of the sample. Thesepreparative reactions generally include, e.g., sample extraction, PCRamplification, nucleic acid fragmentation and labeling, extensionreactions, transcription reactions and the like.

Following sample preparation, the sample can be subjected to one or moredifferent analysis operations. A variety of analysis operations maygenerally be performed, including size based analysis using, e.g.,microcapillary electrophoresis, and/or sequence based analysis using,e.g., hybridization to an oligonucleotide array. In addition to thevarious reaction chambers, the device will generally comprise a seriesof fluid channels which allow for the transportation of the sample or aportion thereof, among the various reaction chambers. Further chambersand components may also be included to provide reagents, buffers, samplemanipulation, e.g., mixing, pumping, fluid direction (i.e., valves)heating and the like.

II. Integratable Operations

A. Sample Acquisition

The sample collection portion of the device of the present inventiongenerally provides for the identification of the sample, whilepreventing contamination of the sample by external elements, orcontamination of the environment by the sample. Generally, this iscarried out by introducing a sample for analysis, e.g., preamplifiedsample, tissue, blood, saliva, etc., directly into a sample collectionchamber within the device. Typically, the prevention ofcross-contamination of the sample may be accomplished by directlyinjecting the sample into the sample collection chamber through asealable opening, e.g., an injection valve, or a septum. Generally,sealable valves are preferred to reduce any potential threat of leakageduring or after sample injection. Alternatively, the device may beprovided with a hypodermic needle integrated within the device andconnected to the sample collection chamber, for direct acquisition ofthe sample into the sample chamber. This can substantially reduce theopportunity for contamination of the sample.

In addition to the foregoing, the sample collection portion of thedevice may also include reagents and/or treatments for neutralization ofinfectious agents, stabilization of the specimen or sample, pHadjustments, and the like. Stabilization and pH adjustment treatmentsmay include, e.g., introduction of heparin to prevent clotting of bloodsamples, addition of buffering agents, addition of protease or nucleaseinhibitors, preservatives and the like. Such reagents may generally bestored within the sample collection chamber of the device or may bestored within a separately accessible chamber, wherein the reagents maybe added to or mixed with the sample upon introduction of the sampleinto the device. These reagents may be incorporated within the device ineither liquid or lyophilized form, depending upon the nature andstability of the particular reagent used.

B. Sample Preparation

In between introducing the sample to be analyzed into the device, andanalyzing that sample, e.g., on an oligonucleotide array, it will oftenbe desirable to perform one or more sample preparation operations uponthe sample. Typically, these sample preparation operations will includesuch manipulations as extraction of intracellular material, e.g.,nucleic acids from whole cell samples, viruses and the like,amplification of nucleic acids, fragmentation, transcription, labelingand/or extension reactions. One or more of these various operations maybe readily incorporated into the device of the present invention.

C. DNA Extraction

For those embodiments where whole cells, viruses or other tissue samplesare being analyzed, it will typically be necessary to extract thenucleic acids from the cells or viruses, prior to continuing with thevarious sample preparation operations. Accordingly, following samplecollection, nucleic acids may be liberated from the collected cells,viral coat, etc., into a crude extract, followed by additionaltreatments to prepare the sample for subsequent operations, e.g.,denaturation of contaminating (DNA binding) proteins, purification,filtration, desalting, and the like.

Liberation of nucleic acids from the sample cells or viruses, anddenaturation of DNA binding proteins may generally be performed byphysical or chemical methods. For example, chemical methods generallyemploy lysing agents to disrupt the cells and extract the nucleic acidsfrom the cells, followed by treatment of the extract with chaotropicsalts such as guanidinium isothiocyanate or urea to denature anycontaminating and potentially interfering proteins. Generally, wherechemical extraction and/or denaturation methods are used, theappropriate reagents may be incorporated within the extraction chamber,a separate accessible chamber or externally introduced.

Alternatively, physical methods may be used to extract the nucleic acidsand denature DNA binding proteins. U.S. Pat. No. 5,304,487, incorporatedherein by reference in its entirety for all purposes, discusses the useof physical protrusions within microchannels or sharp edged particleswithin a chamber or channel to pierce cell membranes and extract theircontents. Combinations of such structures with piezoelectric elementsfor agitation can provide suitable shear forces for lysis. Such elementsare described in greater detail with respect to nucleic acidfragmentation, below.

More traditional methods of cell extraction may also be used, e.g.,employing a channel with restricted cross-sectional dimension whichcauses cell lysis when the sample is passed through the channel withsufficient flow pressure. Alternatively, cell extraction and denaturingof contaminating proteins may be carried out by applying an alternatingelectrical current to the sample. More specifically, the sample of cellsis flowed through a microtubular array while an alternating electriccurrent is applied across the fluid flow. A variety of other methods maybe utilized within the device of the present invention to effect celllysis/extraction, including, e.g., subjecting cells to ultrasonicagitation, or forcing cells through microgeometry apertures, therebysubjecting the cells to high shear stress resulting in rupture.

Following extraction, it will often be desirable to separate the nucleicacids from other elements of the crude extract, e.g., denaturedproteins, cell membrane particles, salts, and the like. Removal ofparticulate matter is generally accomplished by filtration, flocculationor the like. A variety of filter types may be readily incorporated intothe device. Further, where chemical denaturing methods are used, it maybe desirable to desalt the sample prior to proceeding to the next step.Desalting of the sample, and isolation of the nucleic acid may generallybe carried out in a single step, e.g., by binding the nucleic acids to asolid phase and washing away the contaminating salts or performing gelfiltration chromatography on the sample, passing salts through dialysismembranes, and the like. Suitable solid supports for nucleic acidbinding include, e.g., diatomaceuos earth, silica (i.e., glass wool), orthe like. Suitable gel exclusion media, also well known in the art, mayalso be readily incorporated into the devices of the present invention,and is commercially available from, e.g., Pharmacia and Sigma Chemical.

The isolation and/or gel filtration/desalting may be carried out in anadditional chamber, or alternatively, the particular chromatographicmedia may be incorporated in a channel or fluid passage leading to asubsequent reaction chamber. Alternatively, the interior surfaces of oneor more fluid passages or chambers may themselves be derivatized toprovide functional groups appropriate for the desired purification,e.g., charged groups, affinity binding groups and the like, i.e., poly-Toligonucleotides for mRNA purification.

Alternatively, desalting methods may generally take advantage of thehigh electrophoretic mobility and negative of DNA compared to otherelements. Electrophoretic methods may also be utilized in thepurification of nucleic acids from other cell contaminants and debris.In one example, a separation channel or chamber of the device is fluidlyconnected to two separate “field” channels or chambers havingelectrodes, e.g., platinum electrodes, disposed therein. The two fieldchannels are separated from the separation channel using an appropriatebarrier or “capture membrane” which allows for passage of currentwithout allowing passage of nucleic acids or other large molecules. Thebarrier generally serves two basic functions: first, the barrier acts toretain the nucleic acids which migrate toward the positive electrodewithin the separation chamber; and second, the barriers prevent theadverse effects associated with electrolysis at the electrode fromentering into the reaction chamber (e.g., acting as a salt junction).Such barriers may include, e.g., dialysis membranes, dense gels, PEIfilters, or other suitable materials. Upon application of an appropriateelectric filed, the nucleic acids present in the sample will migratetoward the positive electrode and become trapped on the capturemembrane. Sample impurities remaining free of the membrane are thenwashed from the chamber by applying an appropriate fluid flow. Uponreversal of the voltage, the nucleic acids are released from themembrane in a substantially purer form. The field channels may bedisposed on the same or opposite sides or ends of a separation chamberor channel, and may be used in conjucton with mixing elements describedherein, to ensure maximal efficiency of operation. Further, coarsefilters may also be overlaid on the barriers to avoid any fouling of thebarriers by particulate matter, proteins or nucleic acids, therebypermitting repeated use.

In a similar aspect, the high electrophoretic mobility of nucleic acidswith their negative charges, may be utilized to separate nucleic acidsfrom contaminants by utilizing a short column of a gel or otherappropriate matrix or gel which will slow or retard the flow of othercontaminants while allowing the faster nucleic acids to pass.

For a number of applications, it may be desirable to extract andseparate messenger RNA from cells, cellular debris, and othercontaminants. As such, the device of the present invention may, in somecases, include an mRNA purification chamber or channel. In general, suchpurification takes advantage of the poly-A tails on mRNA. In particularand as noted above, poly-T oligonucleotides may be immobilized within achamber or channel of the device to serve as affinity ligands for mRNA.Poly-T oligonucleotides may be immobilized upon a solid supportincorporated within the chamber or channel, or alternatively, may beimmobilized upon the surface(s) of the chamber or channel itself.Immobilization of oligonucleotides on the surface of the chambers orchannels may be carried out by methods described herein including, e.g.,oxidation and silanation of the surface followed by standard DMTsynthesis of the oligonucleotides.

In operation, the lysed sample is introduced into this chamber orchannel in a high salt solution to increase the ionic strength forhybridization, whereupon the mRNA will hybridize to the immobilizedpoly-T. Hybridization may also be enhanced through incorporation ofmixing elements, also as described herein. After enough time has elapsedfor hybridization, the chamber or channel is washed with clean saltsolution. The mRNA bound to the immobilized poly-T oligonucleotides isthen washed free in a low ionic strength buffer. The surface area uponwhich the poly-T oligonucleotides are immobilized may be increasedthrough the use of etched structures within the chamber or channel,e.g., ridges, grooves or the like. Such structures also aid in theagitation of the contents of the chamber or channel, as describedherein. Alternatively, the poy-T oligonucleotides may be immobilizedupon poroussurfaces, e.g., porous silicon, zeolites silica xerogels,scintered particle, or other solid supports.

D. Amplification and In Vitro Transcription

Following sample collection and nucleic acid extraction, the nucleicacid portion of the sample is typically subjected to one or morepreparative reactions. These preparative reactions include in vitrotranscription, labeling, fragmentation, amplification and otherreactions. Nucleic acid amplification increases the number of copies ofthe target nucleic acid sequence of interest. A variety of amplificationmethods are suitable for use in the methods and device of the presentinvention, including for example, the polymerase chain reaction methodor (PCR), the ligase chain reaction (LCR), self sustained sequencereplication (3SR), and nucleic acid based sequence amplification(NASBA).

The latter two amplification methods involve isothermal reactions basedon isothermal transcription, which produce both single stranded RNA(ssRNA) and double stranded DNA (dsDNA) as the amplification products ina ratio of approximately 30 or 100 to 1, respectively. As a result,where these latter methods are employed, sequence analysis may becarried out using either type of substrate, i.e., complementary toeither DNA or RNA.

In particularly preferred aspects, the amplification step is carried outusing PCR techniques that are well known in the art. See PCR Protocols:A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J.and White, T., eds.) Academic Press (1990), incorporated herein byreference in its entirety for all purposes. PCR amplification generallyinvolves the use of one strand of the target nucleic acid sequence as atemplate for producing a large number of complements to that sequence.Generally, two primer sequences complementary to different ends of asegment of the complementary strands of the target sequence hybridizewith their respective strands of the target sequence, and in thepresence of polymerase enzymes and nucleoside triphosphates, the primersare extended along the target sequence. The extensions are melted fromthe target sequence and the process is repeated, this time with theadditional copies of the target sequence synthesized in the precedingsteps. PCR amplification typically involves repeated cycles ofdenaturation, hybridization and extension reactions to producesufficient amounts of the target nucleic acid. The first step of eachcycle of the PCR involves the separation of the nucleic acid duplexformed by the primer extension. Once the strands are separated, the nextstep in PCR involves hybridizing the separated strands with primers thatflank the target sequence. The primers are then extended to formcomplementary copies of the target strands. For successful PCRamplification, the primers are designed so that the position at whicheach primer hybridizes along a duplex sequence is such that an extensionproduct synthesized from one primer, when separated from the template(complement), serves as a template for the extension of the otherprimer. The cycle of denaturation, hybridization, and extension isrepeated as many times as necessary to obtain the desired amount ofamplified nucleic acid.

In PCR methods, strand separation is normally achieved by heating thereaction to a sufficiently high temperature for a sufficient time tocause the denaturation of the duplex but not to cause an irreversibledenaturation of the polymerase enzyme (see U.S. Pat. No. 5,965,188,incorporated herein by reference). Typical heat denaturation involvestemperatures ranging from about 80° C. to 105° C. for times ranging fromseconds to minutes. Strand separation, however, can be accomplished byany suitable denaturing method including physical, chemical, orenzymatic means. Strand separation may be induced by a helicase, forexample, or an enzyme capable of exhibiting helicase activity. Forexample, the enzyme RecA has helicase activity in the presence of ATP.The reaction conditions suitable for strand separation by helicases areknown in the art (see Kuhn Hoffman-Berling, 1978, CSH-QuantitativeBiology, 43:63-67; and Radding, 1982, Ann. Rev. Genetics 16:405-436,each of which is incorporated herein by reference). Other embodimentsmay achieve strand separation by application of electric field acrossthe sample. For example, Published PCT application Ser. Nos. WO 92/04470and WO 92/25177, incorporated herein by reference, describeelectrochemical methods of denaturing double stranded DNA by applicationof an electric field to a sample containing the DNA. Structures forcarrying out this electrochemical denaturation include a workingelectrode, counter electrode and reference electrode arranged in apotentiostat arrangement across a reaction chamber (See, Published PCTapplication Ser. Nos. WO 92/04470 and WO 95/25177, each of which isincorporated herein by reference for all purposes). Such devices may bereadily miniaturized for incorporation into the devices of the presentinvention utilizing the microfabrication techniques described herein.

Template-dependent extension of primers in PCR is catalyzed by apolymerizing agent in the presence of adequate amounts of at least 4deoxyribonucleotide triphosphates (typically selected from dATP, dGTP,dCTP, dUTP and dTTP) in a reaction medium which comprises theappropriate salts, metal cations, and pH buffering system. Reactioncomponents and conditions are well known in the art (See PRC Protocols:A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J.and White, T., eds.) Academic Press (1990), previously incorporated byreference). Suitable polymerizing agents are enzymes known in catalyzetemplate-dependent DNA synthesis.

Published PCT application Ser. No. WO 94/05414, to Northrup and White,discusses the use of a microPCR chamber which incorporates microheatersand micropumps in the thermal cycling and mixing during the PCRreactions.

The amplification reaction chamber of the device may comprise a sealableopening for the addition of the various amplification reagents. However,in preferred aspects, the amplification chamber will have an effectiveamount of the various amplification reagents described above,predisposed within the amplification chamber, or within an associatedreagent chamber whereby the reagents can be readily transported to theamplification chamber upon initiation of the amplification operation. By“effective amount” is meant a quantity and/or concentration of reagentsrequired to carry out amplification of a targeted nucleic acid sequence.These amounts are readily determined from known PCR protocols. See e.g.,Sambrook, et al. Molecular Cloning: A Laboratory Manual, (2nd ed.) Vols.1-3, Cold Spring Harbor Laboratory, (1989) and PCR Protocols: A Guide toMethods and Applications (Innis, M., Gelfand, D., Sninsky, J. and White,T., eds.) Academic Press (1990), both of which are incorporated hereinby reference for all purposes in their entirety. For those embodimentswhere the various reagents are predisposed within the amplification oradjacent chamber, it will often be desirable for these reagents to belyophilized forms, to provide maximum shelf life of the overall device.Introduction of the liquid sample to the chamber then reconstitutes thereagents in active form, and the particular reactions may be carriedout.

In some aspects, the polymerase enzyme may be present within theamplification chamber, couples to a suitable solid support, or to thewalls and surfaces of the amplification chamber. Suitable sold supportsinclude those that are well known in the art, e.g., agarose, cellulose,silica, divinylbenzene, polystyrene, etc. Coupling of enzymes to solidsupports has been reported to impart stability to the enzyme inquestion, which allows for storage of days, weeks or even months withouta substantial loss in enzyme activity, and without the necessity oflyophilizing the enzyme. The 94 kd, single subunit DNA polymerase fromThermus aquaticus (or taq polymerase) is particularly suited for the PCRbased amplification methods used in the present invention, and isgenerally commercially available from, e.g., Promega, Inc., Madison,Wis. In particular, monoclonal antibodies are available which bind theenzyme without affecting its polymerase activity. Consequently, covalentattachment of the active polymerase enzyme to a solid support, or thewalls of the amplification chamber can be carried out by using theantibody as a linker between the enzyme and the support.

In addition to PCR to IVT reactions, the methods and devices of thepresent invention are also applicable to a number of other reactiontypes, e.g., reverse transcription, nick translation, and the like.

E. Labeling and Fragmentation

The nucleic acids in a sample will generally be labeled to facilitatedetection in subsequent steps. Labeling may be carried out during theamplification, in vitro transcription or nick translation processes. Inparticular, amplification, in vitro transcription or nick translationmay incorporate a label into the amplified or transcribed sequence,either through the use of labeled primers or the incorporation oflabeled dNTPs into the amplified sequence.

Alternatively, the nucleic acids in the sample may be labeled followingamplification. Post amplification labeling typically involves thecovalent attachment of a particular detectable group upon the amplifiedsequences. Suitable labels or detectable groups include a variety offluorescent or radioactive labeling groups well known in the art. Theselabels may also be coupled to the sequences using methods that are wellknown in the art. See, e.g., Sambrook, et al.

In addition, amplified sequences may be subjected to other postamplification treatments. For example, in some cases, it may bedesirable to fragment the sequence prior to hybridization with anoligonucleotide array, in order to provide segments which are morereadily accessible to the probes, which avoid looping and/orhybridization to multiple probes. Fragmentation of the nucleic acids maygenerally be carried out by physical, chemical or enzymatic methods thatare known in the art. These additional treatments may be performedwithin the amplification chamber, or alternatively, may be carried outin a separate chamber. For example, physical fragmentation methods mayinvolve moving the sample containing the nucleic acid over pits orspikes in the surface of a reaction chamber or fluid channel. The motionof the fluid sample, in combination with the surface irregularitiesproduces a high shear rate, resulting in fragmentation of the nucleicacids. In one aspect, this may be accomplished in a miniature device byplacing a piezoelectric element, e.g., a PZT ceramic element adjacent toa substrate layer that covers a reaction chamber or flow channel, eitherdirectly, or through a liquid layer, as described herein. The substratelayer has pits, spikes or apertures manufactured in the surface whichare within the chamber or flow channel. By driving the PZT element inthe thickness mode, a standing wave is set up within the chamber.Cavitation and/or streaming within the chamber results in substantialshear. Similar shear rates may be achieved by forcing the nucleic acidcontaining fluid sample through restricted size flow passages, e.g.,apertures having a cross-sectional dimension in the micron or submicronscale, thereby producing a high shear rate and fragmenting the nucleicacid.

A number of sample preparation operations may be carried out byadjusting the pH of the sample, such as cell lysis, nucleic acidfragmentation, enzyme denaturation and the like. Similarly, pH controlmay also play a role in a wide variety of other reactions to be carriedout in the device, i.e., for optimizing reaction conditions,neutralizing acid or base additions, denaturing exogenously introducedenzymes, quenching reactions, and the like. Such pH monitoring andcontrol may be readily accomplished using well known methods. Forexample, pH may be monitored by incorporation of a pH sensor orindicator within a particular chamber. Control may then be carried outby titration of the chamber contents with an appropriate acid or base.

In an alternative aspect, the device may include an electronicallycontrolled pH system. In operation, an electrode is place adjacent,e.g., in fluid contact, to a reaction chamber wehile a counter electrodeis positioned within a second chamber or channel fluidly connected tothe first. Upon application of current to these electrodes, the pH ofthe reaction chamber is altered through the electrolysis of water at thesurface of the electrode, producing O₂ and hydrogen. A pH sensor mayalso be included within the reaction chamber to provide for monitoringand/or feedback control of the precise pH within the chamber.

One example of a reaction chamber employing an electronic pH controlsystem is shown in FIG. 11. As shown, a device 1100 fabricated from twoplanar members 1102 and 1104, includes three distinct chambers, areference chamber 1106, a reaction chamber 1108, and a counter-electrodechamber 1110. Each of the reference chamber 1106 and counter-electrodechamber 1108, e.g., via fluid passages 1112 and 1114. These passages aretypically blocked by an appropriate barrier 1116, e.g., dialysismembrane, gel plug or the like, to prevent the electrophoretic passageof sample elements between the chambers. The reference chamber 1106typically includes a reference electrode 1118. The reference electrodemay be fabricated, e.g., from a platinum, gold or nickel screen pressedwith a mixture of teflon and platinum black (producing a hydrogenelectrode). The reaction chamber 1108 typically includes an electrolysiselectrode 1120, e.g., a platinum, gold or nickel screen coated with anappropriate barrier, e.g., polyacrylamide gel layer, and a hydrogenelectrode 1122, also protected with an appropriate barrier. Thereference electrode 1118 and hydrogen electrode 1122 are connected to anelectrometer 1126 for monitoring the pH within the reaction chamber. Thecounter-electrode chamber 1110 typically includes the counter-electrode1123, e.g., a single platinum, gold or nickel screen electrode. Theelectrolysis electrode and counter-electrode are connected to anappropriate current cource 1124.

Upon introduction of the sample, e.g., a cell suspension or nucleic acidcontaining sample, a current is applied by the current source.Electrolysis at the electrolysis electrode alters the pH within thereaction chamber 1108. The electrometer compares the pH sensed by thevoltage between the reference and hydrogen electrodes. This signal maybe compared to a set-point by appropriate means, e.g., an appropriatelyprogrammed computer or other microprocessor 1128, and used to controlthe application of current. The resulting system allows the automatedcontrol of pH within the reaction chamber by varying the set-pointsignal.

F. Sample Analysis

Following the various sample preparation operations, the sample willgenerally be subjected to one or more analysis operations. Particularlypreferred analysis operations include, e.g., sequence based analysesusing an oligonucleotide array and/or size based analyses using, e.g.,microcapillary array electrophoresis.

1. Oligonucleotide Probe Array

In one aspect, following sample preparation, the nucleic acid sample isprobed using an array of oligonucleotide probes. Oligonucleotide arraysgenerally include a substrate having a large number of positionallydistinct oligonucleotide probes attached to the substrate. Theseoligonucleotide arrays, also described as “Genechip™ arrays,” have beengenerally described in the art, for example, U.S. Pat. No. 5,143,854 andPCT patent publication Ser. Nos. WO 90/15070 and 92/10092. Thesepioneering arrays may be produced using mechanical or light directedsynthesis methods which incorporate a combination of photolithographicmethods and solid phase oligonucleotide synthesis methods. See Fodor etal., Science, 251:767-777 (1991), Pirrung et al., U.S. Pat. No.5,143,854 (see also PCT application Ser. No. WO 90/15070) and Fodor etal., PCT Publication Ser. No. WO92/10092, all incorporated herein byreference. These references disclose methods of forming vast arrays ofpeptides, oligonucleotides and other polymer sequences using, forexample light-directed synthesis techniques. Techniques for thesynthesis of these arrays using mechanical synthesis strategies aredescribed in, e.g., PCT Publication Ser. No. 93/09668 and U.S. Pat. No.5,384,261, each of which is incorporated herein by reference in itsentirety for all purposes. Incorporation of these arrays in injectionmolded polymeric casings has been described in Published PCT applicationSer. No. 95/33846.

The basic strategy for light directed synthesis of oligonucleotidearrays is as follows. The surface of a solid support, modified withphotosensitive protecting groups is illuminated through aphotolithographic mask, yielding reactive hydroxyl groups in theilluminated regions. A selected nucleotide, typically in the form of a3′-O-phosphoramidite-activated deoxynucleoside (protected at the 5′hydroxyl with a photosensitive protecting group), is then presented tothe surface and coupling occurs at the sites that were exposed to light.Following capping and oxidation, the substrate is rinsed and the surfaceis illuminated through a second mask, to expose additional hydroxylgroups for coupling. A second selected nucleotide (e.g., 5′-protected,3′-O-phosphoramidite-activated deoxynucleoside) is presented to thesurface. The selective deprotection and coupling cycles are repeateduntil the desired set of products is obtained. Since photolithography isused, the process can be readily miniaturized to generate high densityarrays of oligonucleotide probes. Furthermore, the sequence of theoligonucleotides at each site is known. See, Pease, et al. Mechanicalsynthesis methods are similar to the light directed methods exceptinvolving mechanical direction of fluids for deprotection and additionin the synthesis steps.

Typically, the arrays used in the present invention will have a sitedensity of greater than 100 different probes per cm². Preferably, thearrays will have a site density of greater than 500/cm², more preferablygreater than about 1000/cm², and most preferably, greater than about10,000/cm². Preferably, the arrays will have more than 100 differentprobes on a single substrate, more preferably greater than about 1000different probes still more preferably, greater than about 10,000different probes and most preferably, greater than 100,000 differentprobes on a single substrate.

For some embodiments, oligonucleotide arrays may be prepared having allpossible probes of a given length. Such arrays may be used in such areasas sequencing or sequence checking applications, which offer substantialbenefits over traditional methods. The use of oligonucleotide arrays insuch applications is described in, e.g., U.S. patent application Ser.No. 08/505,919, filed Jul. 24, 1995, now abandoned, and U.S. patentapplication Ser. No. 08/284,064, filed Aug. 2, 1994, now abandoned, eachof which is incorporated herein by reference in its entirety for allpurposes. These methods typically use a set of short oligonucleotideprobes of defined sequence to search for complementary sequences on alonger target strand of DNA. The hybridization pattern of the targetsequence on the array is used to reconstruct the target DNA sequence.Hybridization analysis of large numbers of probes can be used tosequence long stretches of DNA.

One strategy of de novo sequencing can be illustrated by the followingexample. A 12-mer target DNA sequence is probed on an array having acomplete set of octanucleotide probes. Five of the 65,536 octamer probeswill perfectly hybridize to the target sequence. The identity of theprobes at each site is known. Thus, by determining the locations atwhich the target hybridizes on the array, or the hybridization pattern,one can determine the sequence of the target sequence. While thesestrategies have been proposed and utilized in some applications, therehas been difficulty in demonstrating sequencing of larger nucleic acidsusing these same strategies. Accordingly, in preferred aspects, SBHmethods utilizing the devices described herein use data from mismatchedprobes, as well as perfectly matching probes, to supply useful sequencedata, as described in U.S. patent application Ser. No. 08/505,919, nowabandoned, incorporated herein by reference.

While oligonucleotide probes may be prepared having every possiblesequence of length n, it will often be desirable in practicing thepresent invention to provide an oligonucleotide array which is specificand complementary to a particular nucleic acid sequence. For example, inparticularly preferred aspects, the oligonucleotide array will containoligonucleotide probes which are complementary to specific targetsequences, and individual or multiple mutations of these. Such arraysare particularly useful in the diagnosis of specific disorders which arecharacterized by the presence of a particular nucleic acid sequence. Forexample, the target sequence may be that of a particular exogenousdisease causing agent, e.g., human immunodeficiency virus (see, U.S.application Ser. No. 08/284,064, now abandoned, previously incorporatedherein by reference), or alternatively, the target sequence may be thatportion of the human genome which is known to be mutated in instances ofa particular disorder, i.e., sickle cell anemia (see, e.g., U.S.application Ser. No. 08/082,937, now abandoned, previously incorporatedherein by reference) or cystic fibrosis.

In such an application, the array generally comprises at least four setsof oligonucleotide probes, usually from about 9 to about 21 nucleotidesin length. A first probe set has a probe corresponding to eachnucleotide in the target sequence. A probe is related to itscorresponding nucleotide by being exactly complementary to a subsequenceof the target sequence that includes the corresponding nucleotide. Thus,each probe has a position, designated an interrogation position, that isoccupied by a complementary nucleotide to the corresponding nucleotidein the target sequence. The three additional probe sets each have acorresponding probe for each probe in the first probe set, butsubstituting the interrogation position with the three othernucleotides. Thus, for each nucleotide in the target sequence, there arefour corresponding probes, one from each of the probe sets. The threecorresponding probes in the three additional probe sets are identical tothe corresponding probe from the first probe or a subsequence thereofthat includes the interrogation position, except that the interrogationposition is occupied by a different nucleotide in each of the fourcorresponding probes.

Some arrays have fifth, sixth, seventh and eighth probe sets. The probesin each set are selected by analogous principles to those for the probesin the first four probe sets, except that the probes in the fifth,sixth, seventh and eighth sets exhibit complementarity to a secondreference sequence. In some arrays, the first set of probes iscomplementary to the coding strand of the target sequence while thesecond set is complementary to the noncoding strand. Alternatively, thesecond reference sequence can be a subsequence of the first referencesequence having a substitution of at least one nucleotide.

In some applications, the target sequence has a substituted nucleotiderelative to the probe sequence in at least one undetermined position,and the relative specific binding of the probes indicates the locationof the position and the nucleotide occupying the position in the targetsequence.

Following amplification and/or labeling, the nucleic acid sample isincubated with the oligonucleotide array in the hybridization chamber.Hybridization between the sample nucleic acid and the oligonucleotideprobes upon the array is then detected, using, e.g., epifluorescenceconfocal microscopy. Typically, sample is mixed during hybridization toenhance hybridization of nucleic acids in the sample to nucleoc acidprobes on the array. Again, mixing may be carried out by the methodsdescribed herein, e.g., through the use of piezoelectric elements,electrophoretic methods, or physical mixing by pumping fluids into andout of the hybridization chamber, i.e., into an adjoining chamber.Generally, the detection operation will be performed using a readerdevice external to the diagnostic device. However, it may be desirablein some cases, to incorporate the data gathering operation into thediagnostic device itself.

The hybridization data is next analyzed to determine the presence orabsence of a particular sequence within the sample, or by analyzingmultiple hybridizations to determine the sequence of the target nucleicacid using the SBH techniques already described.

In some cases, hybridized oligonucleotides may be labeled followinghybridization. For example, wghere biotin labeled dNTPs are used in,e.g., amplification or transcription, streptavidin linked reportergroups may be used to label hybridized complexes. Such operations arereadily integratable into the systems of the present invention.

2. Capillary Electrophoresis

In some embodiments, it may be desirable to provide an additional, oralternative means for analyzing the nucleic acids from the sample. Inone embodiment, the device of the invention will optionally oradditionally comprise a micro capillary array for analysis of thenucleic acids obtained from the sample.

Microcapillary array electrophoresis generally involves the use of athin capillary or channel which may or may not be filled with aparticular separation medium. Electrophoresis of a sample through thecapillary provides a size based separation profile for the sample. Theuse of microcapillary electrophoresis in size separation of nucleicacids has been reported in, e.g., Woolley and Mathies, Prod. Nat'l Acad.Sci. USA (1994) 91:11348-11352Microcapillary array electrophoresisgenerally provides a rapid method for size based sequencing, PCR productanalysis and restriction fragment sizing. The high surface to volumeratio of these capillaries allows for the application of higher electricfields across the capillary without substantial thermal variation acrossthe capillary, consequently allowing for more rapid separations.Furthermore, when combined with confocal imaging methods, these methodsprovide sensitivity in the range of attomoles, which is comparable tothe sensitivity of radioactive sequencing methods.

Microfabrication of microfluidic devices including microcapillaryelectrophoretic devices has been discussed in detail in, e.g., Jacobsen,et al., Anal. Chem. (1994) 66:1114-1118, Effenhauser, et al., Anal.Chem. (1994) 66:2949-2953, Harrison, et al, Science (1993) 261:895-897,Effenhauser, et al. Anal. Chem. (1993) 65:2637-2642, and Manz, et al.,J.Chromatog. (1992) 593:263-258. Typically, these methods comprisephotolithographic etching of micron scale channels on a silica, siliconor other rigid substrate or chip, and can be readily adapted for use inthe miniaturized devices of the present invention. In some embodiments,the capillary arrays may be fabricated from the same polymeric materialdescribed for the fabrication of the body of the device, using theinjection molding techniques described herein. In such cases, thecapillary and other fluid channels may be molded into a first planarelement. A second thin polymeric member having ports corresponding tothe termini of the capillary channels disposed therethrough, islaminated or sonically welded onto the first to provide the top surfaceof these channels. Electrodes for electrophoretic control are disposedwithin these ports/wells for application of the electrical current tothe capillary channels. Through use of a relatively this sheet as thecovering member of the capillary channels, heat generated duringelectrophoresis can be rapidly dissipated. Additionally, the capillarychannels may be coated with more thermally conductive material, e.g.,glass or ceramic, to enhance heat dissipation.

In many capillary electrophoresis methods, the capillaries, e.g., fusedsilica capillaries or channels etched, machined or molded into planarsubstrates, are filled with an appropriate separation/sieving matrix.Typically, a variety of sieving matrices are known in the art may beused in the microcapillary arrays. Examples of such matrices include,e.g., hydroxyethyl cellulose, polyacrylamide, agarose and the like. Gelmatrices may be introduced and polymerized within the capillary channel.However, in some cases, this may result in entrapment of bubbles withinthe channels which can interfere with sample separations. Accordingly,it is often desirable to place a preformed separation matrix within thecapillary channel(s), prior to mating the planar elements of thecapillary portion. Fixing the two parts, e.g., through sonic welding,permanently fixes the matrix within the channel. Polymerization outsideof the channels helps to ensure that no bubbles are formed. Further, thepressure of the welding process helps to ensure a void-free system.Generally, the specific gel matrix, running buffers and runningconditions are selected to maximize the separation characteristics ofthe particular application, e.g., the size of the nucleic acidfragments, the required resolution, and the presence of native orundenatured nucleic acid molecules. For example, running buffers mayinclude denaturants, chaotropic agents such as urea or the like, todenature nucleic acds in the sample.

In addition to its use in nucleic acid “fingerprinting” and other sizedbased analyses, the capillary arrays may also be used in sequencingapplications. In particular, gel based sequencing techniques may bereadily adapted for capillary array electrophoresis. For example,capillary electrophoresis may be combined with the Sanger dideoxy chaintermination sequencing methods as discussed in Sambrook, et al. (Seealso Brenner, et al., Proc. Nat'l Acad. Sci. (1989) 86:8902-8906). Inthese methods, the sample nucleic acid is amplified in the presence offluorescent dideoxynucleoside triphosphates in an extension reaction.The random incorporation of the dideoxynucleotides terminatestranscription of the nucleic acid. This results in a range oftranscription products differing from another member by a single base.Comparative size based separation then allows the sequence of thenucleic acid to be determined based upon the last dideoxy nucleotide tobe incorporated.

G. Data Gathering and Analysis

Gathering data from the various analysis operations, e.g.,oligonucleotide and/or microcapillary arrays, will typically be carriedout using methods known in the art. For example, the arrays may bescanned using lasers to excite fluorescently labeled targets that havehybridized to regions of probe arrays, which can then be imaged usingcharged coupled devices (“CCDs”) for a wide field scanning of the array.Alternatively, another particularly useful method for gathering datafrom the arrays is through the use of laser confocal microscopy whichcombines the ease and speed of a readily automated process with highresolution detection. Particularly preferred scanning devices aregenerally described in, e.g., U.S. Pat. Nos. 5,143,854 and 5,424,186.

Following the data gathering operation, the data will typically bereported to a data analysis operation. To facilitate the sample analysisoperation, the data obtained by the reader from the device willtypically be analyzed using a digital computer. Typically, the computerwill be appropriately programmed for receipt and storage of the datafrom the device, as well as for analysis and reporting of the datagathered, i.e., interpreting fluorescence data to determine the sequenceof hybridizing probes, normalization of background and single basemismatch hybridizations, ordering of sequence data in SBH applications,and the like, as described in, e.g., U.S. patent application Ser. No.08/327/525, filed Oct. 21, 1994, now U.S. Pat. No. 5,295,716, andincorporated herein by reference.

III. The Nucleic Acid Diagnostic System

A. Analytical System

A schematic of a representative analytical system based upon the deviceof the invention is shown in FIG. 1. The system includes the diagnosticdevice 2 which performs one or more of the operations of samplecollection, preparation and/or analysis using, e.g., hybridizationand/or size based separation. The diagnostic device is then placed in areader device 4 to detect the hybridization and or separationinformation present on the device. The hybridization and/or separationdata is then reported from the reader device to a computer 6 which isprogrammed with appropriate software for interpreting the data obtainedby the reader device from the diagnostic device. Interpretation of thedata from the diagnostic device may be used in a variety of ways,including nucleic acid sequencing which is directed toward a particulardisease causing agent, such as viral or bacterial infections, e.g.,AIDS, malaria, etc., or genetic disorders, e.g., sickle cell anemia,cystic fibrosis, Fragile X syndrome, Duchenne muscular dystrophy, andthe like. Alternatively, the device can be employed in de novosequencing applications to identify the nucleic acid sequence of apreviously unknown sequence.

B. The Diagnostic Device

1. Generally

As described above, the device of the present invention is generallycapable of carrying out a number of preparative and analytical reactionson a sample. To achieve this end, the device generally comprises anumber of discrete reaction, storage and/or analytical chambers disposedwithin a single unit or body. While referred to herein as a “diagnosticdevice,” those of skill in the art will appreciate that the device ofthe invention will have a variety of applications outside the scope ofdiagnostics, alone. Such applications include sequencing applications,sample identification and characterization applications (for, e.g.,taxonomic studies, forensic applications, i.e., criminal investigations,and the like).

Typically, the body of the device defines the various reaction chambersand fluid passages in which the above described operations are carriedout. Fabrication of the body, and thus the various chambers and channelsdisposed within the body may generally be carried out using one or acombination of a variety of well known manufacturing techniques andmaterials. Generally, the material from which the body is fabricatedwill be selected so as to provide maximum resistance to the full rangeof conditions to which the device will be exposed, e.g., extremes oftemperature, salt, pH, application of electric fields and the like, andwill also be selected for compatibility with other materials used in thedevice. Additional components may be later introduced, as necessary,into the body. Alternatively, the device may be formed from a pluralityof distinct parts that are later assembled or mated. For example,separate and individual chambers and fluid passages may be assembled toprovide the various chambers of the device.

As a miniaturized device, the body of the device will typically beapproximately 1 to 20 cm in length by about 1 to 10 cm in width by about0.1 to about 2 cm thick. Although indicative of a rectangular shape, itwill be readily appreciated that the devices of the invention may beembodied in any number of shapes depending upon the particular need.Additionally, these dimensions will typically vary depending upon thenumber of operations to be performed by the device, the complexity ofthese operations and the like. As a result, these dimensions areprovided as a general indication of the size of the device. The numberand size of the reaction chambers included within the device will alsovary depending upon the specific application for which the device is tobe used. Generally, the device will include at least two distinctreaction chambers, and preferably, at least three, four or five distinctreaction chambers, all integrated within a single body. Individualreaction chambers will also vary in size and shape according to thespecific function of the reaction chamber. For example, in some cases,circular reaction chambers may be employed. Alternatively, elongatereaction chambers may be used. In general however, the reaction chamberswill be from about 0.05 to about 20 mm in width or diameter, preferablyfrom about 0.1 or 0.5 to about 20 mm in width or diameter and about 0.05to about 5 mm deep, and preferably 0.05 to about 1 mm deep. For elongatechambers, length will also typically vary along these same ranges, Fluidchannels, on the other hand, are typically distinguished from chambersin having smaller dimensions relative to the chambers, and willtypically range from about 10 to about 1000 μm wide, preferably, 100 to500 μm wide and about 1 to 500 μm deep. Although described in terms ofreaction chambers, it will be appreciated that these chambers mayperform a number of varied functions, e.g., as storage chambers,incubation chambers, mixing chambers and the like.

In some cases, a separate chamber or chambers may be used as volumetricchambers, e.g., to precisely measure fluid volumes for introduction intoa subsequent reaction chamber. In such cases, the volume of the chamberwill be dictated by volumetric needs of a given reaction. Further, thedevice may be fabricated to include a range of volumetric chambershaving varied, but known volumes or volume ratios (e.g., in comparisonto a reaction chgamber or other volumetric chambers).

As described above, the body of the device is generally fabricated usingone or more of a variety of methods and materials suitable formicrofabrication techniques. For example, in preferred aspects, the bodyof the device may comprise a number of planar members that mayindividually be injection molded parts fabricated from a variety ofpolymeric materials, or may be silicon, glass, or the like. In the caseof substrates like silica, glass or silicon, methods for etching,milling, drilling, etc., may be used to produce wells and depressionswhich make up the various reaction chambers and fluid channels withinthe device. Microfabrication techniques, such as those regularly used inthe semiconductor and microelectronics industries are particularlysuited to these materials and methods. These techniques include, e.g.,electrodeposition, low-pressure vapor deposition, photolithography, wetchemical etching, reactive ion etching (RIE), laser drilling, and thelike. Where these methods are used, it will generally be desirable tofabricate the planar members of the device from materials similar tothose used in the semiconductor industry, i.e., silica, silicon, galliumarsenide, polyimide substrates. U.S. Pat. No. 5,252,294, to Kroy, etal., incorporated herein by reference in its entirety for all purposes,reports the fabrication of a silicon based multiwell apparatus forsample handling in biotechnology applications.

Photolithographic methods of etching substrates are particularly wellsuited for the microfabrication of these substrates and are well knownin the art. For example, the first sheet of a substrate may be overlaidwith a photoresist. An electromagnetic radiation source may then beshone through a photolithographic mask to expose the photoresist in apattern which reflects the pattern of chambers and/or channels on thesurface of the sheet. After removing the exposed photoresist, theexposed substrate may be etched to produce the desired wells andchannels. Generally preferred photoresists include those usedextensively in the semiconductor industry. Such materials includepolymethyl methacrylate (PMMA) and its derivatives, and electron beamresists such as poly(olefin sulfones) and the like (more fully discussedin, e.g., Ghandi, “VLSI Fabrication Principles,” Wiley (1983) Chapter10, incorporated herein by reference in its entirety for all purposes).

As an example, the wells manufactured into the surface of one planarmember make up the various reaction chambers of the device. Channelsmanufactured into the surface of this or another planar member make upfluid channels which are used to fluidly connect the various reactionchambers. Another planar member is then placed over and bonded to thefirst, whereby the wells in the first planar member define cavitieswithin the body of the device which cavities are the various reactionchambers of the device. Similarly, fluid channels manufactured in thesurface of one planar member, when covered with a second planar memberdefine fluid passages through the body of the device. These planarmembers are bonded together or laminated to produce a fluid tight bodyof the device. Bonding of the planar members of the device may generallybe carried out using a variety of methods known in the art and which mayvary depending upon the materials used. For example, adhesives maygenerally be used to bond the planar members together. Where the planarmembers are, e.g., glass, silicon or combinations thereof, thermalbonding, anodic/electrostatic or silicon fusion bonding methods may beapplied. For polymeric parts, a similar variety of methods may beemployed in coupling substrate parts together, e.g., heat with pressure,solvent based bonding. Generally, acoustic welding techniques aregenerally preferred. In a related aspect, this adhesive tapes may beemployed as one portion of the device forming a thin wall of thereaction chamber/channel structures.

Although primarily described in terms of producing a fully integratedbody of the device, the above described methods can also be used tofabricate individual discrete components of the device which are laterassembled into the body of the device.

In additional embodiments, the body may comprise a combination ofmaterials and manufacturing techniques described above. In some cases,the body may include some parts of injection molded plastics, and thelike, while other portions of the body may comprise etched silica orsilicon planar members, and the like. For example, injection moldingtechniques may be used to form a number of discrete cavities in a planarsurface which define the various reaction chambers, whereas additionalcomponents, e.g., fluid channels, arrays, etc, may be fabricated on aplanar glass, silica or silicon chip or substrate. Lamination of one setof parts to the other will then result in the formation of the variousreaction chambers, interconnected by the appropriate fluid channels.

In particularly preferred embodiments, the body of the device is madefrom at least one injection molded, press molded or machined polymericpart that has one or more wells or depressions manufactured into itssurface to define several of the walls of the reaction chamber orchambers. Molds or mold faces for producing these injection molded partsmay generally be fabricated using the methods described herein for,e.g., silicon molds. Examples of suitable polymers for injection moldingor machining include, e.g., polycarbonate, polystyrene, polypropylene,polyethylene, acrylic, and commercial polymers such as Kapton, Valox,Teflon, ABS, Delrin and the like. A second part that is similarly planarin shape is mated to the surface of the polymeric part to define theremaining wall of the reaction chamber(s). Published PCT Application No.95/33846, incorporated herein by reference, describes a device that isused to package individual oligonucleotide arrays. The device includes ahybridization chamber disposed within a planar body. The chamber isfluidly connected to an inlet port and an outlet port via flow channelsin the body of the device. The body includes a plurality of injectionmolded planar parts that are mated to form the body of the device, andwhich define the flow channels and hybridization chamber.

The surfaces of the fluid channels and reaction chambers which contactthe samples and reagents may also be modified to better accommodate adesired reaction. Surfaces may be made more hydrophobic or morehydrophilic depending upon the particular application. Alternatively,surfaces may be coated with any number of materials in order to make theoverall system more compatible to the reactions being carried out. Forexample, in the case of nucleic acid analyses, it may be desireable tocoat the surfaces with, e.g., a teflon or other non-stick coating, toprevent adhesion of nucleic acids to the surface. Additionally,insulator coatings may also be desirable in those instances whereelectrical leads are placed in contact with fluids, to prevent shortingout, or excess gas formation from electrolysis. Such insulators mayinclude those well known in the art, e.g., silicon oxide, ceramics orthe like. Additional surface treatments are described in greater detailbelow.

FIGS. 2A and 2B show a schematic representation of one embodiment of areaction chamber for inclusion in the device of the invention. Thereaction chamber includes a machined or injection molded polymeric part102 which has a well 104 manufactured, i.e., machined or molded, intoits surface. This well may be closed at the end opposite the wellopening as shown in FIG. 2A, or optionally, may be supplied with anadditional opening 118 for inclusion of an optional vent, as shown inFIG. 2B.

The reaction chamber is also provided with additional elements fortransporting a fluid sample to and from the reaction chamber. Theseelements include one or more fluid channels (122 and 110 in FIGS. 2A and2B, respectively) which connect the reaction chamber to an inlet/outletport for the overall device, additional reaction chambers, storagechambers or one or more analytical chambers.

A second part 124, typically planar in structure, is mated to thepolymeric part to define a closure for the reaction chamber. This secondpart may incorporate the fluid channels, as shown in FIGS. 2B and 2B, ormay merely define a further wall of the fluid channels provided in thesurface of the first polymeric part (not shown). Typically, this secondpart will comprise a series of fluid channels manufactured into one ofits surfaces, for fluidly connecting the reaction chamber to an inletport in the overall device or to another reaction or analytical chamber.Again, this second part may be a second polymeric part made by injectionmolding or machining techniques. Alternatively, this second part may bemanufactured from a variety of other materials, including glass, silica,silicon or other crystalline substrates. Microfabrication techniquessuited for these substrates are generally well known in the art and aredescribed above.

In a first preferred embodiment, the reaction chamber is providedwithout an inlet/outlet valve structure, as shown in FIG. 2A. For theseembodiments, the fluid channels 122 may be provided in the surface ofthe second part that is mated with the surface of the polymeric partsuch that upon mating the second part to the first polymeric part, thefluid channel 122 is fluidly connected to the reaction chamber 104.

Alternatively, in a second preferred embodiment, the reaction chambermay be provided with an inlet/outlet valve structure for sealing thereaction chamber to retain a fluid sample therein. An example of such avalve structure is shown in FIGS. 2B. In particular, the second part 124mated to the polymeric part may comprise a plurality of mated planarmembers, wherein a first planar member 106 is mated with the firstpolymeric part 102 to define a wall of the reaction chamber. The firstplanar member 106 has an opening 108 disposed therethrough, defining aninlet to the reaction chamber. This first planar member also includes afluid channel 110 etched in the surface opposite the surface that ismated with the first polymeric part 102. The fluid channel terminatesadjacent to, but not within the reaction chamber inlet 108. The firstplanar member will generally be manufactured from any of the abovedescribed materials, using the above-described methods. A second planarmember 112 is mated to the first and includes a diaphragm valve 114which extends across the inlet 108 and overlaps with the fluid channel110 such that deflection of the diaphragm results in a gap between thefirst and second planar members, thereby creating a fluid connectionbetween the reaction chamber 104 and the fluid channel 110, via theinlet 108. Deflection of the diaphragm valve may be carried out by avariety of methods including, e.g., application of a vacuum,electromagnetic and/or piezoelectric actuators coupled to the diaphragmvalve, and the like. To allow for a deflectable diaphragm, the secondplanar member will typically be fabricated, at least in part, from aflexible material, e.g., silicon, silicone, latex, mylar, polyimide,Teflon or other flexible polymers. As with the reaction chambers andfluid channels, these diaphragms will also be of miniature scale.Specifically, valve and pump diaphragms used in the device willtypically range in size depending upon the size of the chamber or fluidpassage to which they are fluidly connected. In general, however, thesediaphragms will be in the range of from about 0.5 to about 5 mm forvalve diaphragms, and from 1 to about 20 mm in diameter for pumpingdiaphragms. As shown in FIG. 2B, second part 124 includes an additionalplanar member 116 having an opening 126 for application of pressure ofvacuum for deflection of valve 114.

Where reagents involved in a particular analysis are incompatible withthe materials used to manufacture the device, e.g., silicon, glass orpolymeric parts, a variety of coatings may be applied to the surfaces ofthese parts that contact these reagents. For example, components thathave silicon elements may be coated with a silicon nitride layer or ametallic layer of, e.g., gold or nickel, may be sputtered orelectroplated on the surface to avoid adverse reactions with thesereagents. Similarly, inert polymer coatings, e.g., Teflon and the like,pyraline coatings, or surface silanation modifications may also beapplied to internal surfaces of the chambers and/or channels.

The reaction/storage chamber 104 shown in FIG. 2B is also shown with anoptional vent 118, for release of displaced gas present in the chamberwhen the fluid is introduced. In preferred aspects, this vent may befitted with a gas permeable fluid barrier 120, which permits the passageof gas without allowing for the passage of fluid, e.g., a poorly wettingfilter plug. A variety of materials are suitable for use as poorlywetting filter plugs including, e.g., porous hydrophobic polymermaterials, such as spun fibers of acrylic, polycarbonate, teflon,pressed polypropylene fibers, or any number commercially availablefilter plugs (American Filtrona Corp., Richmond, VA, Gelman Sciences,and the like). Alternatively, a hydrophobic membrane can be bonded overa thru-hole to supply a similar structure. Modified acrylic copolymermembranes are commercially available from, e.g., Gelman Sciences (AnnArbor, MI) and particle-track etched polycarbonate membranes areavailable from Poretics, Inc. (Livermore, Calif.). Venting of heatedchambers may incorporate barriers to evaporation of the sample, e.g., areflux chamber or a mineral oil layer disposed within the chamber, andover the top surface of the sample, to permit the evolution of gas whilepreventing excessive evaporation of fluid from the sample.

As described herein, the overall geometry of the device of the inventionmay take a number of forms. For example, the device may incorporate aplurality of reaction chambers, storage chambers and analyticalchambers, arranged in series, whereby a fluid sample is moved seriallythrough the chambers, and the respective operations performed in thesechambers. Alternatively, the device may incorporate a central fluiddistribution channel or chamber having the variousreaction/storage/analytical chambers arranged around and fluidlyconnected to the central channel or chamber, which central channel orchamber acts as a conduit or hub for sample redistribution to thevarious chambers.

As example of the serial geometry of the device is shown in FIG. 3. Inparticular, the illustrated device includes a plurality ofreaction/storage/analytical chambers for performing a number of theoperations described above, fluidly connected in series.

The schematic representation of the device in FIG. 3 shows a device thatcomprises several reaction chambers arranged in a serial geometry.Specifically, the body of the device 200 incorporates reaction chambers202, 206, 210, 214 and 218. These chambers are fluidly connected inseries by fluid channels 208, 212 and 216, respectively.

In carrying out the various operations outlined above, each of thesereaction chambers is assigned one or more different functions. Forexample, reaction chamber 202 may be a sample collection chamber whichis adapted for receiving a fluid sample, i.e., a cell containing sample.For example, this chamber may include an opening to the outside of thedevice adapted for receipt of the sample. The opening will typicallyincorporate a sealable closure to prevent leakage of the sample, e.g., avalve, check-valve, or septum, through which the sample is introduced orinjected. In some embodiments, the apparatus may include a hypodermicneedle or other sample conduit, integrated into the body of the device.and in fluid connection with the sample collection chamber, for directtransfer of the sample from the host, patient, sample vial or tube, orother origin of the sample to the sample collection chamber.

Additionally, the sample collection chamber may have disposed therein, areagent or reagents for the stabilization of the sample for prolongedstorage, as described above. Alternatively, these reagents may bedisposed within a reagent storage chamber adjacent to and fluidlyconnected with the sample collection chamber.

The sample collection chamber is connected via a first fluid channel 204to second reaction chamber 210 in which the extraction of nucleic acidsfrom the cells within the sample may be performed. This is particularlysuited to analytical operations to be performed where the samplesinclude whole cells. The extraction chamber will typically be connectedto sample collection chamber, however, in some cases, the extractionchamber may be integrated within and exist as a portion of the samplecollection chamber. As previously described, the extraction chamber mayinclude physical and or chemical means for extracting nucleic acids fromcells.

The extraction chamber is fluidly connected via a second fluid channel208, to third reaction chamber 210 in which amplification of the nucleicacids extracted from the sample is carried out. The amplificationprocess beings when the sample is introduced into the amplificationchamber. As described previously, amplification reagents may beexogenously introduced, or will preferably be predisposed within thereaction chamber. However, in alternate embodiments, these reagents willbe introduced to the amplification chamber from an optional adjacentreagent chamber for from an external source through a sealable openingin the amplification chamber.

For PCR amplification methods, denaturation and hybridization cyclingwill preferably be carried out by repeated heating and cooling of thesample. Accordingly, PCR based amplification chambers will typicallyinclude a a temperature controller for heating the reaction to carry outthe thermal cycling. For example, a heating element or temperaturecontrol block may be disposed adjacent the external surface of theamplification chamber thereby transferring heat to the amplificationchamber. In this case, preferred devices will include a thin externalwall for chambers in which thermal control is desired. This thin wallmay be a thin cover element, e.g., polycarbonate sheet, or hightemperature tape, i.e. silicone adhesive on Kapton tape (commerciallyavailable from, e.g., 3M corp.). Micro-scale PCR devices have beenpreviously reported. For example, published PCT Application No. WO94/05414, to Northrup and White reports a miniaturized reaction chamberfor use as a PCR chamber, incorporating microheaters, e.g., resistiveheaters. The high surface area to volume ratio of the chamber allows forvery rapid heating and cooling of the reagents disposed therein.Similarly, U.S. Pat. No. 5,304,487 to Wilding et al., previouslyincorporated by reference, also discusses the use of a microfabricatedPCR device.

In preferred embodiments, the amplification chamber will incorporate acontrollable heater disposed within or adjacent to the amplificationchamber, for thermal cycling of the sample. Thermal cycling is carriedout by varying the current supplied to the heater to achieve the desiredtemperature for the particular stage of the reaction. Alternatively,thermal cycling for the PCR reaction may be achieved by transferring thefluid sample among a number of different reaction chambers or regions ofthe same reaction chamber, having different, although constanttemperatures, or by flowing the sample through a serpentine channelwhich travels through a number of varied temperature ‘zones’. Heatingmay alternatively be supplied by exposing the amplification chamber to alaser or other light or electromagnetic radiation source.

The amplification chamber is fluidly connected via a fluid channel,e.g., fluid channel 212, to an additional reaction chamber 214 which cancarry out additional preparative operations, such as labeling orfragmentation.

A fourth fluid channel 216 connects the labeling or fragmentationchamber to an analytical chamber 218. As shown, the analytical chamberincludes an oligonucleotide array 220 as the bottom surface of thechamber. Analytical chamber 218 may optionally, or additionally comprisea microcapillary electrophoresis device 226 and additional preparativereaction chambers, e.g., 224 for performing, e.g., extension reactions,fluidly connected to, e.g., chamber 210. The analytical chamber willtypically have as at least one surface, a transparent window forobservation or scanning of the particular analysis being performed.

FIGS 4A-C illustrate an embodiment of a microcapillary electrophoresisdevice. In this embodiment, the sample to be analyzed is introduced intosample reservoir 402. This sample reservoir may be a separate chamber,or may be merely a portion of the fluid channel leading from a previousreaction chamber. Reservoirs 404, 406 and 414 are filled withsample/running buffer. FIG. 4A illustrates the loading of the sample byplug loading, where the sample is drawn across the intersection ofloading channel 416 and capillary channel 412, by application of anelectrical current across buffer reservoir 405 and sample reservoir 402.In alternative embodiments, the sample is “stack ” loaded by applying anelectrical current across sample reservoir 402 and waste reservoir 414,as shown in FIG 4B. Following sample loading, an electrical field isapplied across buffer reservoir 404 and waste reservoir 414,electrophoresing the sample through the capillary channel 412. Runningof the sample is shown in FIG. 4C. Although only a single capillary isshown in FIGS. 4A-C, the device of the present invention may typicallycomprise more than one capillary, and more typically, will comprise anarray of four or more capillaries, which are run in parallel.Fabrication of the microcapillary electrophoresis device may generallybe carried using the methods described herein and as described in e.g.,Woolley and Mathies, Proc. Nat'l Acad. Sci. USA 91:11348-11352 (1994),incorporated herein by reference in its entirety for all purposes.Typically, each capillary will be fluidly connected to a separateextension reaction chamber for incorporation of a differentdideoxynucleotide.

An alternate layout of the reaction chambers within the device of theinvention, as noted above, includes a centralized geometry having acentral chamber for gathering and distribution of a fluid sample to anumber of separate reaction/storage/analytical chambers arranged around,and fluidly connected to the central chamber. An example of thiscentralized geometry is shown in FIG. 5. In the particular device shown,a fluid sample is introduced into the device through sample inlet 502,which is typically fluidly connected to a sample collection chamber 504.The fluid sample is then transported to a central chamber 508 via fluidchannel 506. Once within the central chamber, the sample may betransported to any one of a number of reaction/storage/analyticalchambers (510, 512, and 514) which are arranged around and fluidlyconnected to the central chamber. As shown, each of reaction chambers510, 512 and 514, includes a diaphragm 516, 518 and 520, respectively,as shown in FIG. 2B, for opening and closing the fluid connectionbetween the central chamber 508 and the reaction chamber. Additionalreaction chambers may be added fluidly connected to the central chamber,or alternatively, may be connected to any of the above describedreaction chambers.

In certain aspect, the central chamber may have a dual function as botha hub and a pumping chamber. In particular, this central pumping chambercan be fluidly connected to one or more additional reaction and/orstores chamber and one or more analytical chambers. The central pumpingchamber again functions as a hub for the various operations to becarried out by the device as a whole as described above. This embodimentprovides the advantage of a single pumping chamber to deliver a sampleto numerous operation, as well as the ability to readily incorporateadditional sample preparation operations within the device by openinganother valve on the central pumping chamber.

In particular, the central chamber 508 may incorporate a diaphragm pumpas one surface of the chamber, and in preferred aspects, will have azero displacement when the diaphragm is not deflected. The diaphragmpump will generally be similar to the valve structure described abovefor the reaction chamber. For example, the diaphragm pump will generallybe fabricated from any one a variety of flexible materials, e.g.,silicon, latex, teflon, mylar and the like. In particularly preferredembodiments, the diaphragm pump is silicon.

With reference to both FIGS. 5A and 5B, central chamber 508 is fluidlyconnected to sample collection chamber 504, via fluid channel 506. Thesample collection chamber end of fluid channel 506 includes a diaphragmvalve 524 for arresting fluid flow. A fluid sample is typicallyintroduced into sample collection chamber through a sealable opening 502in the body of the device, e.g., a valve or septum. Additionally, samplechamber 504 may incorporate a vent to allow displacement of gas of fluidduring sample introduction.

Once the sample is introduced into the sample collection chamber, it maybe drawn into the central pumping chamber 508 by the operation of pumpdiaphragm 526. Specifically, opening of sample chamber valve 524 opensfluid channel 506. Subsequent pulling or deflection of pump diaphragm526 creates negative pressure within pumping chamber 508, therebydrawing the sample through fluid channel 506 into the central chamber.Subsequent closing of the sample chamber valve 524 and relaxation ofpump diaphragm 526, creates a positive pressure within pumping chamber508, which may be used to deliver the sample to additional chambers inthe device. For example, where it is desired to add specific reagents tothe sample, these reagents may be stores in liquid or solid form withinan adjacent storage chamber 510. Opening valve 516 opens fluid channel528, allowing delivery of the sample into storage chamber 510 uponrelaxation of the diaphragm pump. The operation of pumping chamber mayfurther be employed to mix reagents, by repeatedly pulling and pushingthe sample/reagent mixture to and from the storage chamber. This has theadditional advantage of eliminating the necessity of includingadditional mixing components within the device. Additionalchamber/valve/fluid channel structures may be provided fluidly connectedto pumping chamber 508 as needed to provide reagent storage chambers,additional reaction chambers or additional analytical chambers. FIG. 5Aillustrates an additional reaction/storage chamber 514 and valve 520,fluidly connected to pumping chamber 508 via fluid channel 530. Thiswill typically vary depending upon the nature of the sample to beanalyzed, the analysis to be performed, and the desired samplepreparation operation. Following any sample preparation operation,opening valve 520 and closure of other valves to the pumping chamber,allows delivery of the sample through fluid channels 530 and 532 toreaction chamber 514, which may include an analytical device such as anoligonucleotide array for determining the hybridization of nucleic acidsin the sample to the array, or a microcapillary electrophoresis devicefor performing a size based analysis of the sample.

The transportation of fluid within the device of the invention may becarried out by a number of varied methods. For example, fluid transportmay be affected by the application of pressure differentials provided byeither external or internal sources. Alternatively, internal pumpelements which are incorporated into the device may be used to transportfluid samples through the device.

In a first embodiment, fluid samples are moved from onereaction/storage/analytical chamber to another chamber via fluidchannels by applying a positive pressure differential from theoriginating chamber, the chamber from which the sample is to betransported, to the receiving chamber, the chamber to which the fluidsample is to be transported. In order to apply the pressuredifferentials, the various reaction chambers of the device willtypically incorporate pressure inlets connecting the reaction chamber tothe pressure source (positive or negative). For ease of discussion, theapplication of a negative pressure, i.e., to the receiving chamber, willgenerally be described herein. However, upon reading the instantdisclosure, one of ordinary skill in the art will appreciate thatapplication of positive pressure, i.e., to the originating chamber, willbe as effective, with only slight modifications, which will beillustrated as they arise herein.

In one method, application of the pressure differential to a particularreaction chamber may generally be carried out by selectively loweringthe pressure in the receiving chamber. Selective lowering of thepressure in a particular receiving chamber may be carried out by avariety of methods. For example, the pressure inlet for the reactionchamber may be equipped with a controllable valve structure which may beselectively operated to be opened to the pressure source. Application ofthe pressure source to the sample chamber then forces the sample intothe next reaction chamber which is at a lower pressure.

Typically, the device will include a pressure/vacuum manifold fordirecting an external vacuum source to the variousreaction/storage/analytical chambers. A particularly elegant example ofa preferred vacuum pressure manifold is illustrated in FIGS 6A, 6B and6C.

The vacuum/pressure manifold produces a stepped pressure differentialbetween each pair of connected reaction chambers. For example, assumingambient pressure is defined as having a value of 1, a vacuum is appliedto a first reaction chamber, which may be written 1−3x, where x is anincremental pressure differential. A vacuum of 1−2x is applied to asecond reaction chamber in the series, and a vacuum of 1−x is applied toa third reaction chamber. Thus, the first reaction chamber is at thelowest pressure and the third is at the highest, with the second beingat an intermediate level. All chambers, however, are below ambientpressure, e.g., atmospheric. The sample is drawing into the firstreaction chamber by the pressure differential between ambient pressure(1) and the vacuum applied to the reaction chamber (1−3x), whichdifferential is −3x. The sample does not move to the second reactionchamber due to the pressure differential between the first and secondreaction chambers (1−3x vs. 1−2x, respectively). Upon completion of theoperation performed in the first reaction chamber, the vacuum is removedfrom the first chamber, allowing the first chamber to come to ambientpressure, e.g., 1. The sample is then drawn from the first chamber intothe second by the pressure difference between the ambient pressure ofthe first reaction chamber and the vacuum of the second chamber, e.g., 1vs. 1−2x. Similarly, when the operation to be performed in the secondreaction chamber is completed, the vacuum to this chamber is removed andthe sample moves to the third reaction chamber.

A schematic representation of a pneumatic manifold configuration forcarrying out this pressure differential fluid transport system is shownin FIG. 6A. The pneumatic manifold includes a vacuum source 602 which iscoupled to a main vacuum channel 604. The main vacuum channel isconnected to branch channels 606, 608 and 610, which are in turnconnected to reaction chambers 612, 614 and 616, respectively, whichreaction chambers are fluidly connected, in series. The first reactionchamber in the series 616 typically includes a sample inlet 640 whichwill typically include a sealable closure for retaining he fluid sampleand the pressure within the reaction chamber. Each branch channel isprovided with one or more fluidic resistors 618 and 620 incorporatedwithin the branch channel. These fluidic resistors result in atransformation of the pressure from the pressure/vacuum source, i.e., astep down of the gas pressure or vacuum being applied across theresistance. Fluidic resistors may employ a variety of differentstructures. For example, a narrowing of the diameter or cross-sectionalarea of a channel will typically result in a fluidic resistance throughthe channel. Similarly, a plug within the channel which has one or moreholes disposed therethrough, which effectively narrow the channelthrough which the pressure is applied, will result in a fluidicresistance, which resistance can be varied depending upon the numberand/or size of the holes in the plug. Additionally, the plug may befabricated from a porous material which provides a fluidic resistancethrough the plug, which resistance may be varied depending upon theporosity of the material and/or the number of plugs used. Variations inchannel length can also be used to vary fluidic resistance.

Each branch channel will typically be connected at a pressure node 622to the reaction chamber via pressure inlets 624. Pressure inlets 624will typically be fitted with poorly wetting filter plugs 626, toprevent drawing of the sample into the pneumatic manifold in the case ofvacuum based methods Poorly wetting filter plugs may generally beprepared from a variety of materials know in the art and as describedabove. Each branch channel is connected to a vent channel 628 which isopened to ambient pressure via vent 630. A differential fluidic resistor632 is incorporated into the vent channel 628. The fluidic resistancesupplied by fluidic resistor 632 will be less than fluidic resistancesupplied by fluidic resistor 634 which will be less than fluidicresistance supplied by fluidic resistor 636. As described above, thisdifferential fluidic resistance may be accomplished by varying thediameter of the vent channel, varying the number of channels included ina single vent channel, varying channel length, or providing a plug inthe vent channel having a varied number of holes disposed therethrough.

The varied fluidic resistances for each vent channel will result in avaried level of vacuum being applied to each reaction chamber, where, asdescribed above, reaction chamber 616 may have a pressure of 1−3x,reaction chamber 614 may have a pressure of 1−2x and reaction chamber612 may have a pressure of 1−x. The pressure of a given reaction chambermay be raised to ambient pressure, thus allowing the drawing of thesample into the subsequent chamber, by opening the chamber to ambientpressure. This is typically accomplished by providing a sealable opening638 to ambient pressure in the branch channel. This sealable opening maybe a controllable valve structure, or alternatively, a rupture membranewhich may be pierced at a desired time to allow the particular reactionchamber to achieve ambient pressure, thereby allowing the sample to bedrawn into the subsequent chamber. Piercing of the rupture membrane maybe carried out by the inclusion of solenoid operated pins incorporatedwithin the device, or the devices's base unit (discussed in greaterdetail below). In some cases, it may be desirable to prevent back flowfrom a previous or subsequent reaction chamber which is at a higherpressure. This may be accomplished by equipping the fluid channelsbetween the reaction chambers 644 with one-way check valves. Examples ofone-way valve structures include ball and seat structures, flap valves,duck billed check valves, sliding valve structures, and the like.

A graphical illustration of the pressure profiles between three reactionchambers employing a vacuum based pneumatic manifold is shown in FIG.6C. The solid line indicates the starting pressure of each reactionchamber/pressure node. The dotted line indicates the pressure profileduring operation. The piercing of a rupture membrane results in anincrease in the pressure of the reaction chamber to ambient pressure,resulting in a pressure drop being created between the particularchamber and the subsequent chamber. This pressure drop draws the samplefrom the first reaction chamber to the subsequent reaction chamber.

In a similar aspect, a positive pressure source may be applied to theoriginating chamber to push the sample into subsequent chambers. Apneumatic pressure manifold useful in this regard is shown in FIG. 6B.In this aspect, a pressure source 646 provides a positive pressure tothe main channel 604. Before a sample is introduced to the firstreaction chamber, controllable valve 648 is opened to vent the pressurefrom the pressure source and allow the first reaction chamber in theseries 650 to remain at ambient pressure for the introduction of thesample. Again, the first chamber in the series typically includes asample inlet 640 having a sealable closure 642. After the sample isintroduced into the first reaction chamber 650, controllable valve 648is closed, brining the system up to pressure. Suitable controllablevalves include any number of a variety of commercially availablesolenoid valves and the like. In this application, each subsequentchamber is kept at an incrementally higher pressure by the presence ofthe appropriate fluidic resistors and vents, as described above. A basepressure is applied at originating pressure node 652. When it is desiredto deliver the sample to the second chamber 654, sealable opening 656 isopened to ambient pressure. This allows second chamber 654, to come toambient pressure, allowing the pressure applied at the origin pressurenode 652 to force the sample into the second chamber 654. Thus,illustrated as above, the first reaction chamber 650 is maintained at apressure of 1+x, by application of this pressure at originating pressurenode 652. The second reaction chamber 654 is maintained at pressure 1+2xand the third reaction chamber 658 is maintained at a pressure of 1+3x.Opening sealable valve 656 results in a drop in the pressure of thesecond reaction chamber 654 to 1 (or ambient pressure). The pressuredifferential from the first to the second reaction chamber, x, pushesthe sample from the first to the second reaction chamber and eventuallyto the third. Fluidic resistor 660 is provided between pressure node 662and sealable valve 656 to prevent the escape of excess pressure whensealable valve 656 is opened. This allows the system to maintain apositive pressure behind the sample to push it into subsequent chambers.

In a related aspect, a controllable pressure source may be applied tothe originating reaction vessel to push a sample through the device. Thepressure source is applied intermittently, as needed to move the samplefrom chamber to chamber. A variety of devices may be employed inapplying an intermittent pressure to the originating reaction chamber,e.g., a syringe or other positive displacement pump, or the like.Alternatively, for the size scale of the device, a thermopneumatic pumpmay be readily employed. An example of such a pump typically includes aheating element, e.g., a small scale resistive heater disposed in apressure chamber. Also disposed in the chamber is a quantity of acontrolled vapor pressure fluid, such as a fluorinated hydrocarbonliquid, e.g., fluorinert liquids available from 3M Corp. These liquidsare commercially available having a wide range of available vaporpressures. An increase in the controllable temperature of the heaterincreases pressure in the pressure chamber, which is fluidly connectedto the originating reaction chamber. This increase in pressure resultsin a movement of the sample from one reaction chamber to the next. Whenthe sample reaches the subsequent reaction chamber, the temperature inthe pressure chamber is reduced.

The inclusion of gas permeable fluid barriers, e.g., poorly wettingfilter plugs or hydrophobic membranes, in these devices also permits asensorless fluid direction and control system for moving fluids withinthe device. For example, as described above, such filter plugs,incorporated at the end of a reaction chamber opposite a fluid inletwill allow air or other gas present in the reaction chamber to beexpelled during introduction of the fluid component into the chamber.Upon filling of the chamber, the fluid sample will contact thehydrophobic plug thus stopping net fluid flow. Fluidic resistances, asdescribed previously, may also be employed as gas permeable fluidbarriers, to accomplish this same result, e.g., using fluid passagesthat are sufficiently narrow as to provide an excessive fluidresistance, thereby effectively stopping or retarding fluid flow whilepermitting air or gas flow. Expelling the fluid from the chamber theninvolves applying a positive pressure at the plugged vent. This permitschambers which may be filled with no valve at the inlet, i.e., tocontrol fluid flow into the chamber. In most aspects however, a singlevalve will be employed at the chamber inlet in order to ensure retentionof the fluid sample within the chamber, or to provide a mechanism fordirecting a fluid sample to one chamber of a number of chambersconnected to a common channel.

A schematic representation of a reaction chamber employing this systemis shown in FIG. 12A. In brief, the reaction chamber 1202 includes afluid inlet 1204 which is sealed from a fluid passage 1206 by a valve1208. Typically, this valve can employ a variety of structures, asdescribed herein, but is preferably a flexible diaphragm type valvewhich may be displaced pneumatically, magnetically or electrically. Inpreferred aspects, the valves are controlled pneumatically, e.g., byapplying a vacuum to the valve to deflect the diaphragm away from thevalve seat, thereby creating an opening into adjoining passages. At theend opposite from the inlet, is an outlet vent 1210, and disposed acrossthis outlet vent is a hydrophobic membrane 1212. A number of differentcommercially available hydrophobic membranes may be used as describedherein, including, e.g., Versapore 200 R membranes available from GelmanSciences. Fluid introduced into the reaction chamber fills the chamberuntil it contacts the membrane 1212. Closure of the valve then allowsperformance of reactions within the reaction chamber without influencingor influence from elements outside of the chamber.

In another example, these plugs or membranes may be used for degassingor debubbling fluids within the device. For degassing purposes, forexample, a chamber may be provided with one or more vents or with onewall completely or substantially bounded by a hydrophobic membrane toallow the passage of dissolved or trapped gases. Additionally, vacuummay be applied on the external surface of the membrane to draw gasesfrom the sample fluids. Due to the small cross sectional dimensions ofreaction chambers and fluid passages, elimination of such gases takes ongreater importance, as bubbles may interfere with fluid flow, and/orresult in production of irregular data.

In a related aspect, such membranes may be used for removing bubblespurposely introduced into the device, i.e., for the purpose of mixingtwo fluids which were previously desired to be separated. For example,discrete fluids, e.g., reagents, may be introduced into a single channelor debubbling chamber, separated by a gas bubble which is sufficient toseparete the fluid plugs but not to inhibit fluid flow. These fluidplugs may then be flowed along a channel having a vent disposed therein,which vent includes a hydrophobic membrane. As the fluid plugs flow pastthe membrane, the gas will be expelled across the membrane whereupon thetwo fluids will mix. A schematic illustration of such a debubblingchamber is shown in FIG. 12B.

FIG. 12C shows a schematic illustration of a device employing a fluidflow system which utilizes hydrophobic membrane bound vents for controlof fluid flow. As shown, the device 1250 includes a main channel 1252.The main channel is fluidly connected to a series of separate chambers1254-1260. Each of these fluid connections with the main channel 1252 ismediated (opened or closed) by the inclusion of a separate valve1262-1268, respectively, at the intersection of these fluid connectionswith the main channel. Further, each of the various chambers willtypically include a vent port 1270-1276 to the outside environment,which vent ports will typically be bounded by a hydrophobic or poorlywetting membrane. The basic design of this system is reflected in thedevice schematic shown in FIG. 5, as well, in that it employs a centraldistribution chamber or channel.

In operation, samples or other fluids may be introduced into the mainchannel 1252 via a valved or otherwise sealable liquid inlet 1278 or1280. Application of a positive pressure to the fluid inlet, combinedwith the selective opening of the elastomeric valve at the fluidconnection of a selected chamber with the main channel will force thefluid into that chamber, expelling air or other gases through the ventport at the terminus of the selected chamber, until that vent iscontacted with the fluid, whereupon fluid flow is stopped. The valve tothe selected chamber may then be returned to the closed position to sealthe fluid within the chamber. As described above, the requisite pressuredifferential needed for fluid flow may alternatively or additionallyinvolve the application of a negative pressure at the vent port to whichfluid direction is sought.

As a specific example incorporating the device shown in FIG. 12C, asample introduced into the main channel 1252, is first forced into thedegassing chamber 1254 by opening valve 1262 and applying a positivepressure at inlet port 1278. Once the fluid has filled the degassingchamber, valve 1262 may then be closed. Degassing of the fluid may thenbe carried out by drawing a vacuum on the sample through the hydrophobicmembrane disposed across the vent port 1270. Degassed sample may then bemoved from the degassing chamber 1254 to, e.g., reaction chamber 1256,by opening valves 1262 and 1264, and applying a positive pressure to thedegassing chamber vent port 1270. The fluid is then forced from thedegassing chamber 1254, through main channel 1252, into reaction chamber1256. When the fluid fills the reaction chamber, it will contact thehydrophobic membrane, thereby arresting fluid flow. As shown, the deviceincludes a volumetric or measuring chamber 1258 as well as a storagechamber 1260, including similar valve:vent port arrangements 1266:1274and 1268:1276, respectively. The fluid may then be selectively directedto other chambers as described.

FIG. 12D shows a top view of a portion of an injection molded substratefor carrying out the operations schematically illustrated in FIG. 12C.As shown, this device includes liquid loading chambers 1278 a and 1280 awhich are in fluid communication with the fluid inlets 1278 and 1280(not shown). These fluid inlets may typically be fabricated into theinjection molded portion, e.g., drilled into the loading chamber, orfabricated into an overlaying planar member (not shown). Also includedare reaction chambers 1254, degassing chambers 1256 and 1256 a,measuring chambers 1258, and storage chambers 1260. Each of thesechambers is fluidly connected to main channel 1252.

A number of the operations performed by the various reaction chambers ofthe device require a controllable temperature. For example, PCRamplification, as described above, requires cycling of the sample amonga strand separation temperature, an annealing reaction temperature andan extension reaction temperature. A number of other reactions,including extension, transcription and hybridization reactions are alsogenerally carried out at optimized, controlled temperatures. Temperaturecontrol within the device of the invention is generally supplied by thinfilm resistive heaters which are prepared using methods that are wellknown in the art. For example, these heaters may be fabricated from thinmetal films applied within or adjacent to a reaction chamber using wellknown methods such as sputtering, controlled vapor deposition and thelike. The thin film heater will typically be electrically connected to apower source which delivers a current across the heater. The electricalconnections will also be fabricated using methods similar to thosedescribed for the heaters.

Typically, these heaters will be capable of producing temperatures inexcess of 100 degrees without suffering adverse effects as a result ofthe heating. Examples of resistor heaters include, e.g., the heaterdiscussed in Published PCT Application No. WO 94/05414, laminated thinfilm NiCr/polyimide/copper heaters, as well as graphite heaters. Theseheaters may be provided as a layer on one surface of a reaction chamber,or may be provided as molded or machined inserts for incorporation intothe reaction chambers. FIG. 2B illustrates an example of a reactionchamber 104 having a heater insert 128, disposed therein. The resistiveheater is typically electrically connected to a controlled power sourcefor applying a current across the heater. Control of the power source istypically carried out by an appropriately programmed computer. Theabove-described heaters may be incorporated within the individualreaction chambers by depositing a resistive metal film or insert withinthe reaction chamber, or alternatively, may be applied to the exteriorof the device, adjacent to the particular reaction chamber, whereby theheat from the heater is conducted into the reaction chamber.

Temperature controlled reaction chambers will also typically include aminiature temperature sensor for monitoring the temperature of thechamber, and thereby controlling the application of current across theheater. A wide variety of microsensors are available for determiningtemperatures, including, e.g., thermocouples having a bimetallicjunction which produces a temperature dependent electromotive force(EMF), resistance thermometers which include material having anelectrical resistance proportional to the temperature of the material,thermistors, IC temperature sensors, quartz thermometers and the like.See, Horowitz and Hill, The Art of Electronics, Cambridge UniversityPress 1994 (2nd Ed. 1994). One heater/sensor design that is particularlysuited to the device of the present invention is described in, e.g.,U.S. patent application Ser. No. 08/535,875, filed Sep. 28, 1995, andincorporated herein by reference in its entirety for all purposes.Control of reaction parameters within the reaction chamber, e.g.,temperature, may be carried out manually, but is preferably controlledvia an appropriately programmed computer. In particular, the temperaturemeasured by the temperature sensor and the input for the power sourcewill typically be interfaced with a computer which is programmed toreceive and record this data, i.e., via an analog-digital/digital-analog(AD/DA) converter. The same computer will typically include programmingfor instructing the delivery of appropriate current for raising andlowering the temperature of the reaction chamber. For example, thecomputer may be programmed to take the reaction chamber through anynumber of predetermined time/temperature profiles, e.g., thermal cyclingfor PCR, and the like. Given the size of the devices of the invention,cooling of the reaction chambers will typically occur through exposureto ambient temperature, however additional cooling elements may beincluded if desired, e.g., coolant systems, peltier coolers, waterbaths, etc.

In addition to fluid transport and temperature control elements, one ormore of the reaction chambers of the device may also incorporate amixing function. For a number of reaction chambers, mixing may beapplied merely by pumping the sample back and forth into and out of aparticular reaction chamber. However, in some cases constant mixingwithin a single reaction/analytical chamber is desired, e.g., PCRamplification reactions and hybridization reactions.

In preferred aspects, acoustic mixing is used to mix the sample within agiven reaction chamber. In particular, a PZT element (element composedof lead, zirconium and titanium containing ceramic) is contacted withthe exterior surface of the device, adjacent to the reaction chamber, asshown in FIG. 7A. For a discussion of PZT elements for use in acousticbased methods, see, Physical Acoustics, Principles and Methods, Vol. I,(Mason ed., Academic Press, 1965), and Piezoelectric Technology, Datafor Engineers, available from Clevite Corp. As shown, PZT element 702 iscontacting the external surface 704 of hybridization chamber 706. Thehybridization chamber includes as one internal surface, anoligonucleotide array 708. Application of a current to this elementgenerates sonic vibrations which are translated to the reaction chamberwhereupon mixing of the sample disposed therein occurs. The vibrationsof this element result in substantial convection being generated withinthe reaction chamber. A symmetric mixing pattern generated within amicro reaction chamber incorporating this mixing system is shown FIG.7B.

Incomplete contact (i.e., bonding) of the element to the device mayresult in an incomplete mixing of a fluid sample. As a result, theelement will typically have a fluid or gel layer (not shown) disposedbetween the element 702 and the external surface of the device 704,e.g., water. This fluid layer will generally be incorporated within amembrane, e.g., a latex balloon, having one surface in contact with theexternal surface of the reaction chamber and another surface in contactwith the PZT element. An appropriately programmed computer 714 may beused to control the application of a voltage to the PZT element, via afunction generator 712 and RF amplifier 710 to control the rate and/ortiming of mixing.

In alternate aspects, mixing may be supplied by the incorporation offerromagnetic elements within the device which may be vibrated bysupplying an alternating current to a coil adjacent the device. Theoscillating current creates an oscillating magnetic field through thecenter of the coil which results in vibratory motion and rotation of themagnetic particles in the device, resulting in mixing, either by directconvection or accoustic streaming.

In addition to the above elements, the devices of the present inventionmay include additional components for optimizing sample preparation oranalysis. For example, electrophoretic force may be used to draw targetmolecules into the surface of the array. For example, electrodes may bedisposed or patterned on the surface of the array or on the surfaceopposite the array. Application of an appropriate electric field willeither push or pull the targets in solution onto the array. A variety ofsimilar enhancements can be included without departing from the scope ofthe invention.

Although it may often be desirable to incorporate all of the abovedescribed elements within a single disposable unit, generally, the costof some of these elements and materials from which they are fabricated,may make it desirable to provide a unit that is at least partiallyreusable. Accordingly, in a particularly preferred embodiment, a varietyof control elements for the device, e.g., temperature control, mixingand fluid transport elements may be supplied within a reusablebase-unit.

For example, in a particularly preferred embodiment, the reactionchamber portion of the device can be mated with a reusable base unitthat is adapted to receiving the device. As described, the base unit mayinclude one or more heaters for controlling the temperature withinselected reaction chambers within the device. Similarly, the base unitmay incorporate mixing elements such as those described herein, as wellas vacuum or pressure sources for providing sample mixing andtransportation within the device.

As an example, the base unit may include a first surface having disposedthereon, one or more resistive heaters of the type described above. Theheaters are positioned on the surface of the base unit such that whenthe reaction chamber device is mated to that surface, the heaters willbe adjacent to and preferably contacting the exterior surface of thedevice adjacent to one or more reaction chambers in which temperaturecontrol is desired. Similarly, one or more mixing elements, such as theacoustic mixing elements described above, may also be disposed upon thissurface of the base unit, whereby when mated with the reaction chamberdevice, the mixing elements contact the outer surface of thereaction/storage/analytical chambers in which such mixing is desired.For those reaction chambers in which both mixing and heating aredesired, interspersed heaters and mixers may be provided on the surfaceof the base unit. Alternatively, the base unit may include a secondsurface which contacts the opposite surface of the device from the firstsurface, to apply heating on one exterior surface of the reactionchamber and mixing at the other.

Along with the various above-described elements, the base unit alsotypically includes appropriate electrical connections for linking theheating and mixing elements to an appropriate power source. Similarly,the base unit may also be used to connect the reaction chamber deviceitself to external power sources, pressure/vacuum sources and the like.In particular, the base unit can provide manifolds, ports and electricalconnections which plug into receiving connectors or ports on the deviceto provide power, vacuum or pressure for the various control elementsthat are internal to the device. For example, mating of the device tothe base unit may provide a connection from a vacuum source in the baseunit to a main vacuum manifold manufactured into the device, asdescribed above. Similarly, the base unit may provide electricalconnectors which couple to complementary connectors on the device toprovide electrical current to any number of operations within the devicevia electrical circuitry fabricated into the device. Similarly,appropriate connections are also provided for monitoring variousoperations of the device, e.g., temperature, pressure and the like.

For those embodiments employing a pneumatic manifold for fluid transportwhich relies on the piercing of rupture membranes within the device tomove the sample to subsequent chambers, the base unit will alsotypically include one or more solenoid mounted rupture pins. Thesolenoid mounted rupture pins are disposed within receptacles which aremanufactured into the surface of the base unit, which receptaclescorrespond to positions of the rupture membranes upon the device. Thepins are retained below the surface of the base unit when not inoperation. Activation of the solenoid extends the pin above the surfaceof the base unit, into and through the rupture membrane.

A schematic representation of one embodiment of a base unit is shown inFIG. 8. As shown in FIG. 8, the base unit 800 includes a body structure802 having a mating surface 804. The body structure houses the variouselements that are to be incorporated into the base unit. The base unitmay also include one or more thermoelectric heating/cooling elements 806disposed within the base unit such that when the reaction chambercontianing portion of the apparatus is mated to the mating surface ofthe base unit, the reaction chambers will be in contact or immediatelyadjacent to the heating elements. For those embodiments employing adifferential pressure based system for moving fluids within the device,as described above, the base unit may typically include a pressuresource opening to the mating surface via the pressure source port 810.The base unit will also typically include other elements of thesesystems, such as solenoid 812 driven pins 814 for piercing rupturemembranes. These pins are typically within recessed ports 816 in themating surface 804. The base unit will also typically include mountingstructures on the mating surface to ensure proper mating of the reactionchamber containing portion of the device to the base unit. Such mountingstructures generally include mounting pins or holes (not shown) disposedon the mating surface which correspond to complementary structures onthe reaction chamber containing portion of the device. Mounting pins maybe differentially sized, and/or tapered, to ensure mating of thereaction chamber and base unit in an appropriate orientation.Alternatively, the base unit may be fabricated to include a well inwhich the reaction chamber portion mounts, which well has anonsymetrical shape, matching a nonsymetrical shape of the reactionchamber portion. Such a design is similar to that used in themanufacture of audio tape cassettes and players.

In addition to the above described components, the device of the presentinvention may include a number of other components to further facilitateanalyses. In particular, a number of the operations of sample transport,manipulation and monitoring may be performed by elements external to thedevice, per se. These elements may be incorporated within theabove-described base unit, or may be included as further attachments tothe device and/or base unit. For example, external pumps or fluid flowdevices may be used to move the sample through the various operations ofthe device and/or for mixing, temperature controls may be appliedexternally to the device to maximize individual operations, and valvecontrols may be operated externally to direct and regulate the flow ofthe sample. In preferred embodiments, however, these various operationswill be integrated within the device. Thus, in addition to the abovedescribed components, the integrated device of the invention willtypically incorporate a number of additional components for sampletransporting, direction, manipulation, and the like. Generally, thiswill include a plurality of micropumps, valves, mixers and heatingelements.

Pumping devices that are particularly useful include a variety ofmicromachined pumps that have been reported in the art. For example,suitable pumps include pumps which having a bulging diaphragm, poweredby a piezoelectric stack and two check valves, such as those describedin U.S. Pat. Nos. 5,277,556, 5,271,724 and 5,171,132, or powered by athermopneumatic element, as described in U.S. Pat. No. 5,126,022piezoelectric peristaltic pumps using multiple membranes in series, andthe like. The disclosure of each of these patents is incorporated hereinby reference. Published PCT Application No. WO 94/05414 also discussesthe use of a lamb-wave pump for transportation of fluid in micron scalechannels.

Ferrofluidic fluid transport and mixing systems may also be incorporatedinto the device of the present invention. Typically, these systemsincorporate a ferrofluidic substance which is placed into the apparatus.The ferrofluidic substance is controlled/directed externally through theuse of magnets. In particular, the ferrofluidic substance provides abarrier which can be selectively moved to force the sample fluid throughthe apparatus, or through an individual operation of the apparatus.These ferrofluidic systems may be used for example, to reduce effectivevolumes where the sample occupies insufficient volume to fill thehybridization chamber. Insufficient sample fluid volume may result inincomplete hybridization with the array, and incomplete hybridizationdata. The ferrofluidic system is used to sandwich the sample fluid in asufficiently small volume. This small volume is then drawn across thearray in a manner which ensures the sample contacts the entire surfaceof the array. Ferrofluids are generally commercially available from,e.g., FerroFluidics Inc., New Hampshire.

Alternative fluid transport mechanisms for inclusion within the deviceof the present invention include, e.g. electrohydrodynamic pumps (see,e.g., Richter, et al. 3rd IEEE Workshop on Micro Electro MechanicalSystems, Feb. 12-14, 1990, Napa Valley, USA, and Richter et al., Sensorsand Actuators 29:159-165 (1991), U.S. Pat. No. 5,126,022, each of whichis incorporated herein by reference in its entirety for all purposes).Typically, such pumps employ a series of electrodes disposed across onesurface of a channel or reaction/pumping chamber. Application of anelectric field across the electrodes results in electrophoretic movementof nucleic acids in the sample. Indium-tin oxide films may beparticularly suited for patterning electrodes on substrate surfaces,e.g., a glass or silicon substrate. These methods can also be used todraw nucleic acids onto an array. For example, electrodes may paternedon the surface of an array substrate and modified with suitablefunctional groups for coupling nucleic acids to the surface of theelectrodes. Application of a current betwen the electrodes on thesurface of an array and an opposing electrode results in electrophoreticmovement of the nucleic acids toward the surface of the array.

Electrophoretic pumping by application of transient electric fields canalso be employed to avoid electrolysis at the surface of the electrodeswhile still causing sufficient sample movement. In particular, theelectrophoretic mobility of a nucleic acid is not constant with theelectric field applied. An increase in an electric field of from 50 to400 v/cm results in a 30% increase in mobility of a nucleic acid samplein an acrylamide gel. By applying an oscillating voltage between a pairof electrodes capacitively coupled to the electrolyte, a netelectrophoretic motion can be obtained without a net passage of charge.For example, a high electric field is applied in the forward directionof sample movement and a lower field is then applied in the reversedirection. See, e.g., Luckey, et al., Electrophoresis 14:492-501 (1993).

The above described micropumps may also be used to mix reagents andsamples within the apparatus, by directing a recirculating fluid flowthrough the particular chamber to be mixed. Additional mixing methodsmay also be employed. For example, electrohydrodynamic mixers may beemployed within the various reaction chambers. These mixers typicallyemploy a traveling electric field for moving a fluid into which a chargehas been introduced. See Bart, et al., Sensors and Actuators (1990)A21-A-23:193-197. These mixing elements can be readily incorporated intominiaturized devices. Alternatively, mixing may be carried out usingthermopneumatic pumping mechanism. This typically involves the inclusionof small heaters, disposed behind apertures within a particular chamber.When the liquid in contact with the heater is heated, it expands throughthe apertures causing a convective force to be introduced into thechamber, thereby mixing the sample. Alternatively, a pumping mechanismretained behind two one way check valves, such as the pump described inU.S. Pat. No. 5,375,979 to Trah, incorporated herein by reference in itsentirety for all purposes, can be employed to circulate a fluid samplewithin a chamber. In particular, the fluid is drawn into the pumpingchamber through a first one-way check valve when the pump is operated inits vacuum or drawing cycle. The fluid is then expelled from the pumpchamber through another one way check valve during the reciprocal pumpcycle, resulting in a circular fluid flow within the reaction chamber.The pumping mechanism may employ any number of designs, as describedherein, i.e., diaphragm, thermal pressure, electrohydrodynamic, etc.

It will typically be desirable to insulate electrical components of thedevice which may contact fluid samples, to prevent electrolysis of thesample at the surface of the component. Generally, any number ofnon-conducting insulating materials may be used for this function,including, e.g., teflon coating, SiO₂, Si₃N₄, and the like. Preferably,insulating layers will be SiO₂, which may generally be sputtered overthe surface of the component to provide an insulating layer.

The device of the present invention will also typically incorporate anumber of microvalves for the direction of fluid flow within the device.A variety of microvalve designs are particularly well suited for theinstant device. Examples of valves that may be used in the device aredescribed in, e.g., U.S. Pat. No. 5,277,556 to van Lintel, incorporatedherein by reference. Preferred valve structures for use in the presentdevices typically incorporate a membrane or diaphragm which may bedeflected onto a valve seat. For example, the electrostatic valves,silicon/aluminum bimetallic actuated valves or thermopneumatic actuatedvalves can be readily adapted for incorporation into the device of theinvention. Typically, these valves will be incorporated within or at oneor both of the termini of the fluid channels linking the variousreaction chambers, and will be able to withstand the pressures orreagents used in the various operations. An illustration of anembodiment of the diaphragm valve/fluid channel construction isillustrated in FIG. 3.

In alternative aspects, fluidic valves may also be employed. Suchfluidic valves typically include a “liquid curtain” which comprises afluid that is immiscible in the aqueous systems used in the device,e.g., silicone oil, ferrofluidic fluids, and the like. In operation, afluidic valve includes a shallow valving channel, e.g. 50 μm deep,disposed transversely across and interrupting a deeper primary channel,e.g., a 200 μm deep channel in a mating planar member. The valvingchannel is connected to at least one oil port. In operation, the valvingchannel is first filled with oil (or other appropriate fluid element),which is drawn into the channel by capillary action. When gas or liquidare forced through the primary channel, the oil, or “fluid curtain”moves aside and allows passage. In the absence of differential pressurealong the primary channel, the oil will return to seal the fluid or gasbehind a vapor barrier. In such cases, these fluidic valves are usefulin the prevention of evaporation of fluid samples or reagents within thedevice. Additionally, in the case of other fluids, e.g., ferrofluids oroils with suspended metallic particles, application of an appropriatemagnetic field at the valve position immobilizes the fluidic valve,thereby resisting fluid passage at pressures greater than 3-5 psi.Similarly, electrorheological effects may also be employed incontrolling these fluidic valves. For example, the oil portion of thefluid valve may have suspended therein appropriate particles having highdielectric constants. Application of an appropriate electric field thenincreases the viscosity of the fluid thereby creating an appropriatebarrier to fluid flow.

The device may also incorporate one or more filters for removing celldebris and protein solids from the sample. The filters may generally bewithin the apparatus, e.g., within the fluid passages leading from thesample preparation/extraction chamber. A variety of well known filtermedia may be incorporated into the device, including, e.g., cellulose,nitrocellulose, polysulfone, nylon, vinyl/acrylic copolymers, glassfiber, polyvinylchloride, and the like. Alternatively, the filter may bea structure fabricated into the device similar to that described in U.S.Pat. No. 5,304,487 to Wilding et al., previously incorporated herein.Similarly, separation chambers having a separation media, e.g., ionexchange resin, affinity resin or the like, may be included within thedevice to eliminate contaminating proteins, etc.

In addition to sensors for monitoring temperature, the device of thepresent invention may also contain one or more sensors within the deviceitself to monitor the progress of one or more of the operations of thedevice. For example, optical sensors and pressure sensors may beincorporated into one or more reaction chambers to monitor the progressof the various reactions, or within flow channels to monitor theprogress of fluids or detect characteristics of the fluids, e.g., pH,temperature, fluorescence and the like.

As described previously, reagents used in each operation integratedwithin the device may be exogenously introduced into the device, e.g.,through sealable openings in each respective chamber. However, inpreferred aspects, these reagents will be predisposed within the device.For example, these reagents may be disposed within the reaction chamberwhich performs the operation for which the reagent will be used, orwithin the fluid channels leading to that reaction chamber.Alternatively, the reagents may be disposed within storage chambersadjacent to and fluidly connected to their respective reaction chambers,whereby the reagents can be readily transported to the appropriatechamber as needed. For example, the amplification chamber will typicallyhave the appropriate reagents for carrying out the amplificationreaction, e.g., primer probe sequences, deoxynucleoside triphosphates(“dNTPs”), nucleic acid polymerases, buffering agents and the like,predisposed within the amplification chamber. Similarly, samplestabilization reagents will typically be predisposed within the samplecollection chamber.

2. Generic Sample Preparation Device

FIG. 13 shows a schematic illustration of a device configuration forperforming sample preparation reactions, generally, utilizing the fluiddirection systems described herein, e.g., employing external pressures,hydrophobic vents and pneumatic valves. In the configuration shown, fourdomains of the device are each addressed by an array of valves, e.g., a10 valve array, with its own common channel. The four domains maygenerally be defined as: (1) reagent storage; (2) reaction; (3) samplepreparation; and (4) post processing, which are fluidicallyinterconnected. The sample preparation domain is typically used toextract and purify nucleic acids from a sample. As shown, included inthe sample preparation domain are 5 reagent inlets that are fluidlyconnected to larger volume storage vessels, e.g., within the base unit.Examples of such reagents for extraction reactions may include, e.g., 4Mguanidine isothiocyanate, 1×TBE and 50:50 EtOH:H₂O. The two reactionchambers may include, e.g., affinity media for purification of nucleicacids such as glass wool, or beads coated with poly-T oligonucleotides.

The storage domain is linked to the sample preparation domain and isused for storage of reagents and mixtures, e.g., PCR mix with FITC-dGTPand dUTP but no template, UNG reaction mix and IVT reaction mix withouttemplate. The reaction domain is also linked to the sample preparationdomain as well as the storage domain and includes a number of reactionchambers (5), measuring chambers (2) and debubbling chambers (1). Bothsample preparation and reaction domains may be addressed by a thermalcontroller, e.g., heaters or thermoelectric heater/cooler.

The post processing domain is typically linked to the reaction domainand includes a number of reagent inlets (5), reaction chambers (2),storage chambers (1) and sample inlets (1). The reagent inlets may beused to introduce buffers, e.g., 6×SSPE or water into the analyticalelement, e.g., an oligonucleotide array.

3. Generic Multiple Parallel System

FIG. 14 is a schematic illustration of a device configuration foraddressing situations where several reactions are to be carried outunder the same thermal conditions, e.g., multiple parallel sampleanalyses, duplicating multiplex PCR by carrying out several PCRreactions with single primer pairs in parallel followed by recombiningthem, or cycle sequencing with a variety of primer pairs and/ortemplates.

In this configuration as shown, two storage domains supply reagents totwo reaction domains, each being addressed by an array of 50 valves. Thereaction and storage arrays each comprise a 4×12 matrix ofreactors/chambers, each from 10 nl to 5 μl in volume. These chambers areaddressed by 4 columns each of pneumatic ports. Two additional arrays of10 valves address a sample preparation and post processing domain. Abank of solenoid valves may be used to drive the pneumatic ports and thevalve arrays.

IV. Applications

The device and system of the present invention has a wide variety ofuses in the manipulation, identification and/or sequencing of nucleicacid samples. These samples may be derived from plant, animal, viral orbacterial sources. For example, the device and system of the inventionmay be used in diagnostic applications, such as in diagnosing geneticdisorders, as well as diagnosing the presence of infectious agents,e.g., bacterial or viral infections. Additionally, the device and systemmay be used in a variety of characterization applications, such asforensic analysis, e.g., genetic fingerprinting, bacterial, plant orviral identification or characterization, e.g., epidemiological ortaxonomic analysis, and the like.

Although generally described in terms of individual devices, it will beappreciated that multiple devices may be provided in parallel to performanalyses on a large number of individual samples. because the devicesare miniaturized, reagent and/or space requirements are substantiallyreduced. Similarly, the small size allows automation of sampleintroduction process using, e.g., robot samplers and the like.

In preferred aspects, the device and system of the present invention isused in the analysis of human samples. More particularly, the device isused to determine the presence or absence of a particular nucleic acidsequence within a particular human sample. This includes theidentification of genetic anomalies associated with a particulardisorder, as well as the identification within a sample of a particularinfectious agent, e.g., virus, bacteria, yeast or fungus.

The devices of the present invention may also be used in de novosequencing applications. In particular, the device may be used insequencing by hybridization (SBH) techniques. The use of oligonucleotidearrays in de novo SBH applications is described, for example, in U.S.application Ser. No. 08/082,937, filed Jun. 25, 1993.

EXAMPLES Example 1 Extraction and Purification of Nucleic Acids

In separate experiments, HIV cloned DNA was spiked into either horseblood or a suspension of murine plasmacytoma fully differentiatedB-cells derived from BALBc mice. Guanidine isothiocyanate was added to aconcentration of 4 M, to lyse the material. In separate experiments, thelysate was passed through a cartridge containing glass wool (20 μl), acartridge with soda glass walls (20 μl), and a glass tube. After 30minutes at room temperature, the remaining lysate was washed away withseveral volumes of ethanol:water (1:1) and the captured DNA was elutedat 60° C. using 1×TBE. The yield of eluted DNA was measured usingethidum bromide staining on an agarose gel, and purity was tested byusing the eluted material as a template for a PCR reaction. Elutionyields ranged from 10% to 25% and PCR yields ranged from 90 to 100% ascompared to controls using pure template.

Example 2 RNA Preparation Reactions in Miniaturized System

A model miniature reactor system was designed to investigate theefficacy of miniaturized devices in carrying out prehybridizationpreparative reactions on target nucleic acids. In particular, a dualreaction chamber system for carrying out in vitro transcription andfragmentation was fabricated. The device employed a tube based structureusing a polymer tubing as an in vitro transcription reactor coupled to aglass capillary fragmentation reactor. Reagents not introduced with thesample were provided as dried deposits on the internal surface of theconnecting tubing. The experiment was designed to investigate theeffects of reaction chamber materials and reaction volume in RNApreparative reaction chambers.

The sample including the target nucleic acid, DNA amplicons containing a1 kb portion of the HIV gene flanked with promoter regions for the T3and T7 RNA primers on the sense and antisense strands, respectively, RNApolymerase, NTPs, fluorinated UTP and buffer, were introduced into thereactor system at one end of the tubing based system. In vitrotranscription was carried out in a silicone tubing reactor immersed in awater bath. Following this initial reaction, the sample was movedthrough the system into a glass capillary reactor which was maintainedat 94° C., for carrying out the fragmentation reaction. The products ofa representative time-course fragmentation reaction are shown in the gelof FIG. 10A. In some cases, the tubing connecting the IVT reactor to thefragmentation reactor contained additional MgCl₂ for addition to thesample. The glass capillary was first coated with BSA to avoidinteractions between the sample and the glass. Following fragmentation,the sample was hybridized with an appropriately tiled oligonucleotidearray, as described above. Preparation using this system with 14 mMMgCl₂ addition resulted in a correct base calling rate of 96.5%.Omission of the MgCl₂ gave a correct base calling rate of 95.5%.

A similar preparative transcription reaction was carried out in amicro-reaction chamber fabricated in polycarbonate. A well was machinedin the surface of a first polycarbonate part. The well was 250 μm deepand had an approximate volume of 5 μl. A second polycarbonate part wasthen acoustically welded to the first to provide a top wall for thereaction chamber. The second part had two holes drilled through it,which holes were positioned at opposite ends of the reaction chamber.Temperature control for the transcription reaction was supplied byapplying external temperature controls to the reaction chamber, asdescribed for the tubing based system. 3 μl samples were used for bothtranscription and fragmentation experiments.

Transcription reactions performed in the micro-reactor achieved a 70%yield as compared to conventional methods, e.g., same volume inmicrofuge tube and water bath or PCR thermal cycler. A comparison of invitro transcription reaction products using a microchamber versus alarger scale control are shown in FIG. 10B.

Example 3 PCR Amplification in Miniaturized System

The miniature polymeric reaction chamber similar to the one described inExample 2 was used for carrying out PCR amplification. In particular,the chamber was fabricated from a planar piece of poycarbonate 4 mmthick, and having a cavity measuring 500 μm deep machined into itssurface. A second planar polycarbonate piece was welded over the cavity.This second piece was only 250 μm thick. Thermal control was supplied byapplying a peltier heater against the thinner second wall of the cavity.

Amplification of a target nucleic acid was performed with Perkin-ElmerGeneAmp® PCR kit. The reaction chamber was cycled for 20 seconds at 94°C. (denaturing), 40 seconds at 65° C. (annealing) and 50 seconds at 72°C. (extension). A profile of the thermal cycling is shown in FIG. 9.Amplification of approximately 10⁹ was shown after 35 cycles. FIG. 10Cshows production of amplified product in the microchamber as compared toa control using a typical PCR thermal cycler.

Example 4 System Demonstration, Integrated Reactions

A microfabricated polycarbonate device was manufactured having thestructure shown in FIG. 15A. The device included three discrete ventedchambers. Two of the chambers (top and middle) were thermally isolatedfrom the PCR chamber (bottom) to prevent any denaturation of the RNApolymerase used in IVT reractions at PCR temperatures. Thermal isolationwas accomplished by fabricating the chambers more than 10 mm apart in athin polycarbonate substrate and controlling the temperatures in eachregion through the use of thermoelectric temperature controllers, e.g.,peltier devices.

The reactor device dimensions were as follows: channels were 250 μm wideby 125 μm deep; the three reaction chambers were 1.5 mm wide by 13 mm inlength by 125 to 500 μm deep, with the reactor volumes ranging from 2.5to 10 μl. Briefly, PCR was carried out by introducing 0.3 units of Taqpolymerase, 0.2 mM dNTPs, 1.5 mM MgCl₂, 0.2 μM primer sequences,approximately 2000 molecules of template sequence and 1×Perkin-Elmer PCRbuffer into the bottom chamber. The thermal cycling program included (1)an initial denaturation at 94° C. for 60 seconds, (2) a denaturationstep at 94° C. for 20 seconds, (3) an annealing step at 65° C. for 40seconds, (4) an extension step at 72° C. for 50 seconds, (5) repeatedcycling through steps 2-4 35 times, and (6) a final extension step at72°°C. for 60 seconds.

Following PCR, 0.2 μl of the PCR product was transferred to the IVTchamber (middle) along with 9.8 μl of IVT mixture (2.5 mM ATP, CTP, GTPand 0.5 mM UTP, 0.25 mM Fluorescein-UTP, 8 mM MgCl₂, 50 mM HEPES,1×Promega Transcription Buffer, 10 mM DTT, 1 unit T3 RNA polymerase, 0.5units RNAguard (Pharmacia)) that had been stored in a storage chamber(top). Fluid transfer was carried out by applying pressure to the ventsat the termini of the chambers. IVT was carried out at 37° C. for 60minutes.

The results of PCR and IVT are shown in FIG. 15B, compared with controlexperiments, e.g., performed in eppendorf tubes.

Example 5 Acoustic Mixing

The efficacy of an acoustic element for mixing the contents of areaction chamber was tested. A 0.5″×0.5″×0.04″ crystal of PZT-5H wasbonded to the external surface of a 0.030″ thick region of a planarpiece of delrin which had cavity machined in the surface opposite thePZT element. An oligonucleotide array synthesized on a flat silicasubstrate, was sealed over the cavity using a rubber gasket, such thatthe surface of the array having the oligonucleotide probes synthesizedon it was exposed to the cavity, yielding a 250 μl reaction chamber. ThePZT crystal was driven by an ENI200 High Frequency Power Supply, whichis driven by a function generator form Hewlett Packard that was gated bya second function generator operated at 1 Hz.

In an initial test, the chamber was filled with deionized water and asmall amount of 2% milk was injected for visualization. The crystal wasdriven at 2 MHz with an average power of 3 W. Fluid velocities withinthe chamber were estimated in excess of 1 mm/sec, indicating significantconvection. A photograph showing this convection is shown in FIG. 7B.

The efficacy of acoustic mixing was also tested in an actualhybridization protocol. For this hybridization test, a fluorescentlylabeled oligonucleotide target sequence having the sequence5′-GAGATGCGTCGGTGGCTG-3′ and an array having a checkerboard pattern of400 μm squares having complements to this sequence synthesized thereon,were used. Hybridization of a 10 nM solution of the target in 6×SSPE wascarried out. During hybridization, the external surface of the array waskept in contact with a thermoelectric cooler set at 15° C. Hybridizationwas carried out for 20 minutes while driving the crystal at 2 MHz at anaverage power of 4 W (on time=0.2 sec., off time=0.8 sec.). Theresulting average intensity was identical to that achieved usingmechanical mixing of the chamber (vertical rotation with an incorporatedbubble).

Additional experiments using fluorescently labeled and fragmented 1 kbportion of the HIV virus had a successful base calling rates. Inparticular, a 1 kb HIV nucleic acid segment was sequenced using an HIVtiled oligonucleotide array or chip. See, U.S. patent application Ser.No. 08/284,064, filed Aug. 2, 1994, and incorporated herein by referencefor all purposes. Acoustic mixing achieved a 90.5% correct base callingrate as compared to a 95.8% correct base calling rate for mechanicalmixing.

Example 6 Demonstration of Fluid Direction System

A polycarbonate cartridge was fabricated using conventional machining,forming an array of valves linking a common channel to a series ofchannels leading to a series of 10 μl chambers, each of which wasterminated in a hydrophobic vent. The chambers included (1) an inletchamber #1, (2) inlet chamber #2, (3) reaction chamber, (4) debubblingchamber having a hydrophobic vent in the center, (5) a measuring chamberand (6) a storage chamber. Elastomeric valves were opened and closed byapplication of vacuum or pressure (approx. 60 psi) to the space abovethe individual valves.

In a first experiment, water containing blue dye (food coloring) wasintroduced into inlet chamber #1 while water containing yellow dye (foodcoloring) was introduced into inlet chamber #2. By opening theappropriate valves and applying 5 psi to the appropriate vent, thefollowing series of fluid movements were carried out: the blue water wasmoved from inlet chamber #1 to the reaction chamber; the yellow waterwas moved from inlet chamber #2 to the storage chamber #6; the bluewater was moved from the reaction chamber to the measuring chamber andthe remaining blue water was exhausted to the inlet chamber #1; Themeasured blue water (approximately 1.6 μl) was moved from the measuringchamber to the debubbling chamber; the yellow water is then moved fromthe storage chamber into the debubbling chamber whereupon it linked withthe blue water and appeared to mix, producing a green color; andfinally, the mixture was moved from the debubbling chamber to thereaction chamber and then to the storage chamber.

Functioning of the debubbling chamber was demonstrated by moving fourseparate plugs of colored water from the reaction chamber to thedebubbling chamber. The discrete plugs, upon passing into the debubblingchamber, joined together as a single fluid plug.

The functioning of the measuring chamber was demonstrated byrepetetively moving portions of a 10 μl colored water sample from thestorage chamber to the measuring chamber, followed by exhausting thisfluid from the measuring chamber. This fluid transfer was carried out 6times, indicating repeated aliquoting of approximately 1.6 μl permeasuring chamber volume (10 μl in 6 aliquots).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

What is claimed is:
 1. A method of directing a fluid sample in aminiature fluidic system, comprising: providing a microfabricated devicehaving at least first and second chambers disposed therein, wherein eachof said at least first and second chambers is in fluid connection with acommon chamber or channel, has at least first and second controllablevalves disposed across said fluid connection, respectively, and whereinat least one of said first and second chambers include a vent; applyinga positive pressure to said common chamber or channel; selectivelyopening said at least first controllable valve, whereby said positivepressure forces said fluid sample from said common chamber or channelinto said first chamber.
 2. The method of claim 1, further comprisingapplying a positive pressure to said at least first chamber andselectively opening said at least first controllable valve, whereby saidpositive pressure forces said fluid sample from said at least firstchamber into said common chamber or channel.
 3. The method of claim 1,wherein said vent comprises a hydrophobic membrane sealably disposedacross said vent, whereby when said fluid sample contact saidhydrophobic membrane, flowing of said fluid sample into said at leastfirst chamber stops.
 4. The method of claim 1, wherein said at leastfirst and second controllable valves are selectively openedpneumatically.
 5. The method of claim 1, further comprising: degasingthe fluid sample by: closing the at least first controllable valve; andapplying a reduced pressure at said vent.
 6. The method of claim 1,wherein, said first chamber is a volumetric chamber having a knownvolume, such that said volumetric chamber is filled with a first aliquotof said fluid sample, thereby measuring a known volume of said fluidsample.
 7. The method of claim 6, wherein each of said chambers of saiddevice provided in said providing step has a cross sectional dimensionof from about 0.05 to about 20 mm, and a depth dimension of from about0.05 to about 5 mm.
 8. The method of claim 6, further comprising:applying a positive pressure to said at least first chamber andselectively opening said at least first controllable valve, whereby saidpositive pressure forces said fluid sample from said volumetric chamberinto said second chamber.